AU735248B2 - Marsupial contraceptive vaccine - Google Patents

Description

AUSTRALIA

Patents Act 1990 MARSUPIAL CRC LIMITED

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Marsupial contraceptive vaccine The following statement is a full description of this invention including the best method of performing it known to us: 4 R.

Marsupial contraceptive vaccine Field of the Invention The present invention relates to isolated marsupial zona pellucida (ZP2 and ZP3) polypeptides and to polynucleotides encoding these polypeptides. The present invention also relates to a contraceptive vaccine composition for use in a marsupial female and to a method of inhibiting conception in marsupials.

Background of the Invention Mvarsupials present a dichotomy in population management. Despite their status in Australia as protected native animals, and even as national icons, some marsupial species can cause serious economic and environmental problems, and boom and bust population swings raise serious concerns about animal welfare (Caughley et al. 1987; Gibson and Young 1987). Even a highly valued species such as the koala is a candidate for control because unregulated population growth threatens the survival of populations where their local abundance has lead to the death of appropriate food trees (Phillips 1990). The situation in New Zealand is quite different.

Six species of Australian marsupials are now established in New Zealand and, although most pose only local problems, the common brushtail possumr is now the country's number one vertebrate pest with serious impacts on the environment, animal health and ultimately the economy (Cowan 1990).

For Australia and New Zealand in particular.. there is a need to develop humane techniques of population management to achieve damage mitigation, whether the damage is economic, environmental or to animal welfare. In. New Zealand the aim is a generalised reduction in the number of brushtail possums across the country, However, in Australia the aim is not a generalized reduction in the population of a marsupial species nationally because the problems are localised. Any strategy or agent will thus have to be not only specific to the targeted marsupial but will have to have effects :that limit its geographical and temnporal impact so that survival of the species is assured. A related problem is the management of captive populations.

These -not only include animals in zoos and fauna parks but animals whose free movement is restricted either intentionally or unintentionally by security fences or other forms of urban development. Currently, the only management tools available for such populations are the culling of excess aninials or surgical sterilization, usually by vasectomy.

There is growing consensus that the regulation of fertility is a desirable management tool for wild animals, provided that it is effective, humane and poses no significant threat to the environment. The development of a safe and effective vaccine for the control of marsupial populations is therefore desirable.

Contraceptive vaccines are useful in circumstances where relatively long term but not permanent contraception is desired. A contraceptive vaccine preferably should have an effect which is long lasting and highly specific.

Fertilization is a complex interaction between key regulatory molecules on the surface of the sperm and egg, which is poorly understood in all marsupials. The vital role that these molecules play in the reproductive process make them likely targets for control of fertility in many species including the brushtail possum.

All mammalian eggs are surrounded by a zona pellucida, a relatively simple coat composed of three glycoproteins (ZP1, ZP2 and ZP3) that contains the egg's sperm receptor. The glycoprotein components of the zona pellucida serve specific functions during fertilization and early development, S: which are well defined in the mouse. ZP1 is a structural protein that 20 crosslinks filaments of ZP2 and ZP3. During fertilization sperm initially bind to ZP3, which induces the sperm acrosome reaction. After induction of the acrosome, ZP2 acts as a secondary sperm receptor which is necessary for penetration of the sperm through the zona pellucida. Following fertilization the proteolytic cleavage of ZP2 and modification of ZP3 are thought to be involved in the prevention of polyspermy.

The important role of the zona pellucida during fertilization, its unique expression in growing ooctyes and strong immunogenicity has made it an attractive target for the development of immunocontraceptive vaccines (reviewed by Epifano Dean, 1994). Immunization with whole zonae or 30 components of the zona pellucida, has been shown to inhibit fertilization in several species including primates, rabbits, rodents and dogs (reviewed by Prasad et al. 1996).

The genes that code for the zona pellucida proteins have been characterised in several mammalian and other vertebrate species. All of the ZP genes are expressed only in growing oocytes (reviewed by Liang Dean, 1993) and although the biological functions of the zona pellucida exhibit species specificity the genes are conserved among diverse vertebrate species.

The ZP3 gene is the most highly conserved of the three with amino acid identities between eutherian mammals (human, mouse, rabbit, pig) ranging from 66-74 (reviewed by Epifano Dean, 1994). The ZP2 gene is also well conserved with amino acid identities between eutherian mammals (human, mouse, rabbit, pig) ranging from 54-72% (Epifano Dean, 1994). ZP2 homologues (25-30% identity to mouse ZP2) have also been identified in three species of teleost fish (Lyons et al., 1993; Chang et al., 1997) and Xenopus laevis (Hedrick, 1996). There have been no reports to date, however, of cloning or sequencing of a marsupial ZP2 or ZP3 gene.

Summary of the Invention The present inventors have now cloned and sequenced the possum and wallaby ZP3 genes and the possum ZP2 gene.

Accordingly, in a first aspect the present invention provides an isolated polynucleotide, the polynucleotide including a sequence characterised by nucleotides 4 to 2142 of SEQ ID NO:1; (ii) a sequence characterised by nucleotides 1 to 1216 of SEQ ID NO:3; 20 (iii) a sequence characterised by nucleotides 1 to 1269 of SEQ ID NO:5; or e (iv) a sequence which hybridises selectively under stringent conditions to a sequence as defined in any one of paragraphs to (iii) above or a sequence complementary thereto.

By "hybridises selectively" we mean that the sequence hybridises to a sequence defined in any one of paragraphs to (iii) but does not hybridise under stringent conditions to a non-marsupial ZP2 or ZP3 gene.

When used herein, stringent conditions are those that employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO 4 at 50 0 C; employ during hybridisation a denaturing agent such as formamide, for example, (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll. 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaC1, 75 mM sodium citrate at 42 0 C: or employ 50% formamide, 5 x SSC (0.75 M NaC1, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42 0 C in 0.2 x SSC and 0.1% SDS.

In a preferred embodiment, the polynucleotide sequence is less than 5000 nucleotides in length, more preferably less than 2000 nucleotides in length, more preferably less than 1000 nucleotides in length and more preferably less than 500 nucleotides in length.

In a preferred embodiment the isolated polynucleotide has a sequence selected from a sequence characterised by nucleotides 4 to 2142 of SEQ ID NO:1; (ii) a sequence characterised by nucleotides 1 to 1216 of SEQ ID NO:3; (iii) a sequence characterised by nucleotides 1 to 1269 of SEQ ID NO:5; or (iv) an allelic variant or functional equivalent of a sequence as defined in any one of paragraphs to (iii) above.

By "allelic variant" we mean a variant of a sequence defined in any one of paragraphs to (iii) which occurs naturally in a marsupial and which encodes a ZP2 or ZP3 protein. By "functional equivalent" we mean a sequence which differs from a sequence defined in any one of paragraphs (i) to (iii) but which, through the degeneracy of the genetic code, encodes a polypeptide substantially as shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.

In a further preferred embodiment, the variants or functional 25 equivalents of the present invention share at least 50% homology, more preferably at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology and more preferably at least homology with a sequence shown in any one of SEQ ID NOS: 1, 3 or wherein the homology is calculated by the BLAST program blastn as S 30 described in Altschul, Madden, Schaffer, Zhang, Zhang, SZ., Miller, W. And Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Research 25(17):3389-3402.

The polynucleotide of the present invention may comprise DNA or RNA sequences.

In a second aspect the present invention provides a vector including a polynucleotide according to the first aspect of the present invention.

In a third aspect the present invention provides a bacterial, yeast, insect, plant or mammalian host cell transformed with a vector according to the second aspect of the present invention.

In a fourth aspect the present invention provides a method of producing a marsupial ZP2 or ZP3 polypeptide which includes culturing a host cell according to the third aspect of the present invention under conditions enabling the expression of the polypeptide and optionally recovering the polypeptide.

In a fifth aspect the present invention provides a marsupial ZP2 or ZP3 polypeptide produced by a method according to the fourth aspect of the present invention.

In one preferred embodiment of the third and fourth aspects of the present invention, the host cell is a bacterial cell. It has also been recognised, however, that the oligosaccharide residues of the ZP2 and ZP3 proteins play an important role in fertilization. Accordingly, recombinant ZP2 and ZP3 polypeptides may be glycosylated in order to maximise potential immunocontraceptive effects. In another preferred embodiment of 20 the present invention, therefore, recombinant proteins are produced by an expression system which gives rise to glycosylated recombinant ZP2 or ZP3 polypeptides, such as a baculovirus expression system; a Semliki Forest Virus expression system; a stable transformation of a mammalian cell line with ZP2 or ZP3 gene under the control of an inducible promoter; or 25 agrobacterium mediated or direct gene transfer of ZP2 or ZP3 into plants to produce stable transgenics that express recombinant protein either in the whole plant or in a particular tissue or organ.

In a sixth aspect, the present invention provides an oligonucleotide probe or primer of at least 8 nucleotides, the oligonucleotide having a sequence that hybridizes selectively to a polynucleotide of the first aspect of the present invention.

In a further preferred embodiment the oligonucleotide comprises at least 10 nucleotides, more preferably at least 18 nucleotides and more preferably at least 25 nucleotides.

In a further preferred embodiment the oligonucleotide probe is conjugated with a label such as a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.

In a seventh aspect, the present invention provides an isolated polypeptide having a sequence which is: shown in SEQ ID NO: 2; (ii) shown in SEQ ID NO: 4; (iii) shown in SEQ ID NO: 6; or (iv) a derivative or biologically active fragment of a sequence as defined in any one of paragraphs to (iii) above.

By derivative" we mean a sequence derived by insertion, deletion or substitution of amino acids in a sequence defined in any one of paragraphs to (iii), wherein the derivative encodes a polypeptide which retains at least one of the activities of a marsupial ZP2 or ZP3 protein. Such activities include the ability to mimic the binding of a native marsupial ZP2 or ZP3 protein to at least one antibody or ligand molecule.

In a preferred embodiment, the derivative shares at least homology, more preferably at least 70% homology, more preferably at least 80% homology, more preferably at least 90% homology and more preferably at least 95% homology with a sequence shown in any one of SEQ ID NOS: 2, 4 or wherein the homology is calculated by the BLAST program blastx as described in Altschul, Madden, Schaffer, Zhang, Zhang, Miller, W. And Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Research *e 5* 25 25(17):3389-3402.

By "biologically active fragment" we mean a fragment of a sequence shown in any one of SEQ ID NOS: 2, 4 or 6 which retains at least one of the activities of a native marsupial ZP2 or ZP3 proteins which activities include the ability to mimic the binding of a native marsupial ZP2 or ZP3 protein to 30 at least one antibody or ligand molecule.

The present inventors have identified particular fragments in the marsupial ZP2 polypeptide which have potential for use in a contraceptive vaccine. Accordingly, in a preferred embodiment of the seventh aspect of the invention, the biologically active fragment includes a sequence selected from the group consisting of: TSIALQVGSILIAEIFFVAVLCLVKCM (SEQ ID NO: 7): (ii) QRLTLSSRMVCILGPVTCN (SEQ ID NO: 8); (iii) IFRCAHVVTSENLILRATYKSCAERVHGRVHGTYRVNLKFL (SEQ ID NO: 9); (iv) PQVAWTVIVGYS (SEQ ID NO: LYTVALQLTYG (SEQ ID NO: 11); (vi) NVPVNLS (SEQ ID NO: 12); and (vii) TSGVAVE (SEQ ID NO: 13).

The present inventors have also identified a particular biologically active fragment in the marsupial ZP3 polypeptide which has potential for use in a contraceptive vaccine.

Accordingly, in a further preferred embodiment of the seventh aspect of the invention, the biologically active fragment includes a sequence which corresponds to amino acid nos 334 to 348 in SEQ ID NO: 6. In this embodiment the biological fragment has the sequence SLSSSRKRSLADQEP (SEQ ID NO: 14). In another preferred embodiment the fragment corresponds to amino acid nos 316-330 in SEQ ID NO: 4. In this embodiment the fragment has the sequence HLSSSRKRSLANQEP(SEQ ID NO: In an eighth aspect the present invention provides a chimeric polypeptide comprising a first polypeptide, the first polypeptide being a 20 polypeptide according to the seventh aspect of the present invention, and a second polypeptide.

"The second polypeptide preferably facilitates presentation of the first polypeptide for the purpose of raising an immune response against the first polypeptide. Accordingly, the second polypeptide may be any peptide sequence which is suitable for this function. For example, the second polypeptide may be selected from the group consisting of keyhole limpet hemacyanin (KLH), tetanus toxoid thyroglobulin, albumin, chicken lysosyme, haemaglutinin, Hepatitis B antigen, purified protein derivative of tuberculin, cholera toxin, cholera toxin B subuit, diphtheria toxoid, E. Coli 30 enterotoxin, cytokines and E.coli heat labile toxin (LT or LTB). In a preferred embodiment, the second polypeptide is keyhole limpet hemacyanin (KLH) or tetanus toxoid (TT).

It will be appreciated by those skilled in the art that a number of modifications may be made to the polypeptides and fragments of the present invention without deleteriously affecting the biological activity of the polypeptides or fragments. This may be achieved by various changes, such as sulfation, phosphorylation, nitration and halogenation; or by amino acid insertions, deletions and substitutions, either conservative or nonconservative (eg. D-amino acids, desamino acids) in the peptide sequence where such changes do not substantially alter the overall biological activity of the peptide. Preferred substitutions are those which are conservative, i.e., wherein a residue is replaced by another of the same general type. As is well understood, naturally-occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar. Furthermore, three of the encoded amino acids are aromatic. It is generally preferred that encoded peptides differing from the determined polypeptide contain substituted codons for amino acids which are from the same group as that of the amino acid replaced. Thus, in general, the basic amino acids Lys, Arg, and His are interchangeable; the acidic amino acids Asp and Glu are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gin, and Asn are interchangeable; the nonpolar aliphatic amino acids Gly, Ala, Val, Ile, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, Ile and Leu are more closely related), and the aromatic amino acids Phe, Trp and Tyr are interchangeable.

It should further be noted that if polypeptides are made synthetically, 20 substitutions by amino acids which are not naturally encoded by DNA may also be made. For example, alternative residues include the omega amino acids of the formula NH 2

(CH

2 )nCOOH wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine, t-butyl alanine, t-butyl glycine, Nmethyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, 25 Tyr or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.

In a ninth aspect, the present invention provides a composition for use in raising an immune response in animals against the ZP2 or ZP3 30 proteins, the composition including an acceptable carrier and a polypeptide according to the seventh or eighth aspects of the present invention.

In a tenth aspect, the present invention provides a composition for use in raising an immune response in animals against marsupial ZP2 or ZP3 proteins, the composition including an acceptable carrier and a polynucleotide according to the first aspect of the present invention.

9 As will be appreciated by those skilled in the field the nucleic acid sequence can be delivered as naked nucleic acid or using a suitable viral or bacterial vectors or multicellular organisms such as the parasitic nematode (see, for example, Heath, Cowan, Stankiewicz, Clark, Homer, G., Tempero, Jowett, Flanagan, Shubber, Street, McElrea, G., Chilvers, Newton-Howse, Jowett, J. And Morrison, L. (1998) Possum biological control parasites and bacteria, In: "Biological Control of Possums", Ed. R. Lynch, Royal Society of New Zealand, Wellington, NZ, pp13-19, the entire contents of which are incorporated herein by reference). Suitable bacterial vectors include the bacteria Salmonella spp. Suitable viral vectors include, for example, retroviral vectors, adenoviral vectors, poxviruses and vaccinia vectors. An example of a suitable pox virus is ectromelia (see, for example, Jackson, Maguire, Hinds, L.A. and Ramshaw, I.A. (1998) Infertility in mice induced by a recombinant ectromelia virus expressing mouse zona pellucida glycoprotein 3, Biol. Reprod. 58: 152-159, the entire contents of which are incorporated herein by reference). An example of a suitable vaccinia vector is a modified Vaccinia Ankara vector.

Further information regarding nucleic acid vaccines can be found in WO 96/03144 and in Suhrbier A (1997 Multi-epitope DNA vaccines, 20 Immunol Cell Biol 75(4):402-408 the disclosures of which are incorporated herein by cross reference.

A composition of the present invention may be in the form of a live vaccine, such as a replication limited Macropod Herpesvirus vaccine or a Salmonella vaccine (see, for example, Curtiss III, R. (1990) Attenuated 25 Salmonella strains as live vectors for the expression of foreign antigen, In: Woodrow, G.C. and Leviine, M.M. (eds) New Generation Vaccine. Marcel Dekker Inc. New York. Pp 161-188; and Zhang, Lou, Koopman, M., Doggett, Tung, K.S.K. and Curtiss III, R. (1997) Antibody responses and infertility in mice following oral immunisation with attenuated Salmonella S. 30 t!yphimurium expressing recombinant murine ZP3. Biology of Reproduction 56:33-41, the entire contents of which are incorporated herein by reference).

.A composition of the present invention may also be in the form of a bacterial ghost vaccine. Bacterial ghost vaccines are described in Szostak, Hensel, Eko, Klein, Auer, Mader, Haslberger, A., Bunka, Wanner, G. and Lubitz, W. (1996) Bacterial ghosts: non-living candidate vaccines. J. Biotechnol. 44: 161-170, the entire contents of which are incorporated herein by reference.

A composition of the present invention may be in a form suitable for injectable use. Forms suitable for injectable use preferably include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. They are preferably stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms canll be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable 20 compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required followed by 25 filtered sterilization. Generally, dispersions are preparezt by incorporating the various sterilized active ingredients into a sterile vehicle which contains .ooo the basic dispersion medium and other ingredients from those enumerated *above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the 30 freeze-drying technique which yield a powder of the active ingredients plus any additional desired ingredient from previously sterile-filtered solution thereof, When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier. or they may be enclosed in hard or soft shell gelatin capsule, or compressed into tablets, or incorporated directly with the food of the diet.

For oral administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about O.l g and 2000mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like, and a lubricant such as magnesium stearate. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. A syrup or elixir may contain the active 20 compound, methyl and propylparabens as preservatives, and a dye. Of course, any material used in preparing any dosage unit form should be veterinarilly pure and substantially non-toxic in the amounts employed. In addition, the actie compound(s) may be incorporated into sustained-release preparations and formulations.

Acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and .o absorption delaying agents and the like. The use of such media and agents for veterinarilly active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, 30 use thereof in the therapeutic compositions is contemplated. Preferred adjuvants include DEAE Dextran/mineral oil, Alhydrogel, Auspharm adjuvant and Algammulin. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. For administration to marsupials it is particularly advantageous to use a multi- 12 dose container linked to a repeating vaccination gun. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired contraceptive effect in association with the required pharmaceutical carrier.

The principal active ingredient may be compounded for convenient and effective administration in effective amounts with a suitable veterinarilly acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5pg to about 2000mg. Expressed in proportions, the active compound is generally present in from about 0.5pg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

In an eleventh aspect the present invention provides an antibody which binds selectively to a polypeptide of the seventh aspect of the present invention.

By "binds selectively" we mean that the antibody does not bind to a non-marsupial ZP2 or ZP3 protein.

20 The term "antibody" should be construed as covering any specific binding substance having a binding domain with the required specificity.

Thus, the term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide including an immunoglobulin binding domain, whether natural or synthetic. Chimeric 25 molecules including an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.

In a twelfth aspect the present invention provides a method of inhibiting fertilization of an egg by a sperm in a female marsupial which method includes inoculating the female marsupial with a polypeptide 30 according to the seventh aspect of the invention.

In a preferred embodiment of this aspect of the present invention the marsupial is a possum or macropod or other species as listed below in Table 1.

Throughout this specification the word "comprise" or variations thereof will be understood to imply the inclusion of a stated element or integer or group of stated elements or integers but not the exclusion of any other element of integer of group of integers.

Table 1 Marsupial species that are potential targets for immunocontraceptive control Common Name Scientific Name Brushtail possum Koala Eastern grey kangaroo Western grey kangaroo Red Kangaroo Common wallaroo Bennett's (or red necked) wallaby Tammar wallaby Whiptail wallaby Swamp wallaby Agile wallaby 20 Pademelon Trichosurus vulpecula Phascolarctos cinereus Macropus giganteus Macropus fuliginosus Macropus rufus Macropus robustus Macropus rufogriseus Macropus eugenii Macropus parr'n Wallabia bicolor Macropus agilis Thylogale billardierii

S

S..

S

S.

S. S

S

In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following non-limitiing Examples and Figures.

Brief Description of the Figures Figure 1. Serum titre (log10) of porcine ZP antibodies of control and immunised possums. Animals were immunised in weeks 0, 4 and Bars represent standard error of the mean and indicates significant differences between control and immunised groups 0.05).

Detailed Description of the Invention Example 1 Effect of immunisation against porcine ZP on the fertility of female possums The introduced marsupial Brushtail possum (Trichosurus vulpecula) is a major pest species in New Zealand. Research is under way to develop a possum-specific method of immunologically-based fertility control (immunocontraception). Vaccines targeting the zona pellucida (ZP) layer around the egg have been successfully used for contraception in a range of eutherian mammals but have never been tested in a marsupial. This study determined the effect of immunisation against porcine ZP on the fertility of female possums in order to evaluate whole porcine zona pellucida (ZP) as an immunocontraceptive for possums.

Methods Adult female possums were immunised s.c. at time 0 with 1500 porcine ZP using complete Freund's adjuvant and with 1500 ZP (week 4) and 6000 ZP (week 10) using incomplete Freund's adjuvant. Control females (n=12) were treated with the same adjuvants plus 1 mg ovalbumin. Possums :i were established in mating groups (1 immunised 1 control and 1 untreated per group) and the following measurements recorded: serum titre of anti-ZP 20 antibodies binding to porcine ZP using FITC-labelling number of matings between week 2 and week 18 determined by presence of sperm in daily urine samples number of offspring born between week 4 and week 30 number of follicles present 3 days after ovarian stimulation with 10 IU PMISG Results 25 Immunisation against porcine ZP increased anti-porcine

ZP

antibodies in serum (Figure 1) had no effect on mating behaviour but reduced the conception rates of immunised animals between weeks 2 and 18 (Table reduced the proportion of females producing offspring between weeks 4 and 30 3/12 immunised vs 8/12 controls (One-tail Fisher's exact test, 30 p< 0.05); and had no effect on the number of large ovarian follicles promoted by PMSG treatment (Table 3).

Table 2 Number of possums mated and the proportion of animals that conceived between week 2 and week 18 following immunisation against porcine ZP Control Immunised Number of animals 12 12 Number of animals mated 11 11 Total number of matings 16 21 Percentage of fertile matings 37.5% 9.5% p<0.05 Table 3 Number of follicles present (mean sem) in ovaries of control and immunised possum following PMSG stimulation Control Immunised Total follicles 21.0 4.2 17.1 Follicles 1-2 mm 15.6 3.7 11.8 3.1 Follicles 2-3 mm 3.7 0.9 3.0 0.8 Follicles <3 mm 1.7 0.4 2.3 0.6 0 Conclusions :V 406 Prelimihary results indicate that similar to other species, immunisation against porcine ZP reduced the fertility of treated possums, however, in contrast to some eutherian mammals, there was no effect on 25 ovarian function. Antigens in porcine ZP show promise for fertility-based possum control in New Zealand. A possum-specific peptide from possum ZP3 is currently being tested for its immunocontraceptive potential.

Example 2 30 Cloning of the Brushtail possum and wallaby ZP3 genes The initial strategies taken for cloning of marsupial Zp-3 were based on an assumption that there would be quite a high degree of homology between eutherian and marsupial ZP genes. A full length mouse Zp-3 cDNA clone was used for Southern and northern analysis of the possum and wallaby Zp-3 gene and also for screening possum and wallaby ovarian cDNA libraries. Our Southern analysis detected the Zp-3 gene in mouse genomic 16 DNA (which served as a positive control) but did not detect a homologous gene in possum or wallaby genomic DNA. The northern analysis indicated expression of Zp-3 mRNA in mouse ovaries but expression of a homologous gene was not detected in adult possum or wallaby ovaries. Screening of possum and wallaby cDNA libraries on two occasions with mouse Zp-3 cDNA did not result in identification of any positive clones.

A PCR approach based on the conserved regions of the eutherian Zp-3 gene, amplified a product of the expected size from possum ovary which had homology to mouse Zp-3. The PCR product however had a high homology to a mammalian growth arrest-specific protein (gas-6). Another eutherian probe, an anti-porcine ZP antibody raised in rabbit does cross-react with Brushtail possum and tammar wallaby ZP, but when used to probe a possum ovary cDNA library for ZP genes it specifically recognised a clone which had high homology to the human BAP31 gene. These studies demonstrated that cloning of marsupial ZP genes based solely on their homology to the eutherian proteins was not a suitable approach.

The successful cloning strategy involved the alignment of Zp-3 cDNA nucleotide sequences from the mouse, pig and human with a fish vitelline membrane protein to identify the most highly conserved regions within the gene. Several conserved regions were chosen and primers for reverse transcription polymerase chain reaction (RT-PCR) were designed. One set of primers amplified a 440 bp product that was confirmed by sequencing to be a portion of the Zp-3 gene. This PCR product was used to screen possum and wallaby ovary cDNA libraries in order to isolate full length clones.

25 Positive clones were isolated from the libraries and sequenced.

Results and Discussion The full length possum and wallaby Zp-3 genes have been cloned and fully sequenced. The possum ZP3 gene sequence is shown in SEQ ID NO: and the corresponding polypeptide sequence is shown in SEQ ID NO: 6. The wallaby ZP3 gene sequence is shown in SEQ ID NO: 3 and the corresponding polypeptide sequence is shown in SEQ ID NO: 4. The degree of nucleic acid and amino acid sequence identity between Brushtail possum ZP3 and that of wallaby. mouse and pig shown in Table 4. Possum ZP3 is most similar to wallaby ZP3 (89% amino acid identity) and 40-45% similar to most eutherian 35 species. The comparisons between wallaby ZP3 and the other species is probably an over-estimate of the degree of similarity because it is based on a 440 bp PCR product within the most highly conserved region of the gene.

A region of the possum ZP3 protein that has potential as a reproductive vaccine has been identified and synthesised. This region corresponds to amino acid nos 334 to 348 in SEQ ID NO: 6 and has the sequence SLSSSRKRSLADQEP (SEQ ID NO: 14). This peptide was selected as the corresponding region of murine ZP3 contains overlapping T cell and B cell epitopes and greater than 90% of mice immunized with these peptides develop autoimmune oophoritis and become infertile (Rhim et al., 1992).

The effects of immunisation with this peptide are irreversible making it useful for control of pest populations such as the possum. The possum sequence for this peptide has only 2-3 amino acids in common with that of eutherian species and therefore is likely to be marsupial specific. The corresponding fragment in the wallaby sequence, which also has potential as a reproductive vaccine, is amino acid nos 316-330 shown in SEQ ID NO: 4 and has the sequence HLSSSRKRSLANQEP (SEQ ID NO: Table 4: A comparison of possum, wallaby, pig and mouse ZP3 cDNAs and proteins.

Nucleic Acid Identity ZP3 possum wallaby pig mouse possum 92 57 53 Amino wallaby* 89 44 43 Identity mouse 42 40 66 all comparisons based on 400bp of wallaby ZP3 sequence d decrease possum numbers by reducing the breeding success of possuls, may provide an effective, long-term method of population control. The zona 18 pellucida (ZP) is a glycoprotein layer surrounding the egg that must be penetrated by the sperm in order for fertilization to occur (Paterson Aitken, 1990) and is therefore a potential target for contraception.

The sequence of possum ZP3, one of the proteins in the possum ZP has recently been characterised so that its potential as an immunocontraceptive can now be assessed. An important criteria for any immunocontraceptive for possums is that it should not effect the fertility of non-target species. A possum-specific region of the ZP3 molecule that may have potential as a reproductive vaccine has been identified and synthesised.

This region comprises the amino acid sequence SLSSSRKRSLADQEP

(SEQ

ID NO: 14). A short peptide sequence such as this is unlikely to elicit an immune response alone so the ZP3 peptide was bound to carrier molecules.

Two carrier proteins were tested: i) keyhole limpet hemacyanin (KLH) and ii) a peptide sequence from tetanus toxoid (TT) in attempt to maximise the antibody-based and cellular immune responses. The effect of immunisation was studied in both male and female possums. This research will provide important information on the immunogenicity of the ZP3 peptide and it potential for use in fertility control in possums.

In the immunogenicity study possums were immunised subcutaneously as follows: 1. Controls against KLH only (n=4 females 4 males) against TT only (n=4 females 4 males) 2. ZP3 peptide-KLH (n=8 females 4 males) 3. ZP3 peptide-TT (n=8 females 4 males).

25 Initial vaccinations used 300 ug antigen in complete Freund's adjuvant followed by booster vaccination in weeks 4 and 8 using incomplete Freund's adjuvant.

Throughout the 18 week trial, antibodies against ZP3 peptide were high in animals immunised against ZP3 conjugated to either KLH and TT epitope but binding was low in control animals. FITC-labelling indicated that in most treated animals immunisation against ZP3 peptide resulted in the binding of possum immunoglobulin IgG to the ZP of medium and large sized ovarian follicles but was not common in control animals. No differences in the number of large follicles present at the time of post mortem 35 were detected. Histological analysis indicates no difference in total number of follicles or follicular development.

These results indicate that both vaccine preparations were able to stimulate a sustained antibody immune response against the ZP3 peptide and that immunisation was associated with a antibody response presumably to the native ZP in the ovaries of treated possums. As the antibody titre was greatest following treatment with the ZP3-KLH conjugate, this formulation is currently being tested in a breeding trial. The effect of immunisation on ovarian activity, conception rates, pouch young production and follicle development is being studied in female possums.

Example 4 Cloning of the Brushtail possum ZP2 gene A 279 bp fragment of Brushtail possum ZP2 was amplified by reverse transcription followed by polymerase chain reaction (RT-PCR). The nucleotide sequences of ZP2 cDNA from mouse, human, rabbit, Xenopus and carp were extracted from Genbank and aligned to identify the most highly conserved regions of the gene. Primers for PCR were designed within two of the most conserved regions: 5'-GTC ATC TAT GAA AAT GAA-3' (forward) and 5'-CAT GTA AAT TGG TTG GCG GAG-3' (reverse) and subsequently synthesised (Bresatec, Adelaide, Australia). PCR products of the correct size were sequenced by automated sequencing at the Biomolecular Research Facility at the University of Newcastle. Analysis of the nucleotide sequence was carried out using the Australian National Genome Information Service (ANGIS) to confirm that the product amplified was ZP2.

25 Approximately 1 x 106 clones from a Brushtail possum ovary Uni- ZAP cDNA library were screened with the 279 bp Brushtail possum ZP2 PCR product. Positive clones were plaque-purified and plasmids were excision rescued according to the manufacturers protocol (Stratagene). Plasmids were sequenced at the Biomolecular Research Facility at the University of Newcastle and the sequence analysed using ANGIS.

Results and Discussion The full length possum ZP-2 gene has been cloned and fully sequenced. The 2176 nucleotide full-length Brushtail possum ZP2 nucleotide sequence (cDNA) is shown in SEQ ID NO: 1 and the amino acid 35 sequence is shown in SEQ ID NO: 2. The nucleotide sequence has a single open reading frame of 2136 nucleotides flanked by relatively short 5' and 3' untranslated regions of 3 and 37 nucleotides respectively. The ATG initiation codon is incorporated into an ANNATG motif (AAAATG) normally associated with initiator codons in vertebrates, and a AATAAA polyadenylation signal is located 14 nucleotides downstream of the TAA termination codon. The open reading frame encodes a protein of 712 amino acids with a predicted molecular mass of 79,542. A signal sequence cleavage site is predicted at amino acid 41, indicating an N-terminal signal peptide of amino acids. Brushtail possum ZP2 has two transmembrane domains situated toward the carboxyl terminal region of the protein (amino acids 595- 611 and 681-702).

Overall similarity of the ZP2 nucleotide sequence and aligned amino acid residues between the Brushtail possum and other species is summarised in Table 5. The nucleic acid sequence of possum ZP2 is more similar to that of eutherian mammals (60-65%) than that of amphibian or fish (37- Similarly the amino acid identity between Brushtail possum and eutherian mammals (48-54%) is also greater than that between possum and amphibian or fish (approx. 30%) (Table Table 5: A comparison of Brushtail possum (Trichosurus vulpecula) ZP2 nucleotide and amino acid sequence with that of mouse, human, rabbit, cat, Xenopus, white flounder and carp.

Species Identit to Brushtail Possum Amino Acid Nucleotide Mouse 47.8 Human 51.3 63 Rabbit 54.5 at 50.8 63.5 Xenopus 23.6 41 White Flounder* 32.7 37 Carp* 29.5 42 does not include repetitive domain There are 18 cysteine residues present in possum ZP2 that are shared with mouse and human, and there are an additional 4 cysteine residues in possum ZP2 that is not present in any of the other species. Brushtail possum ZP2 contains 9 potential sites for N-linked glycosylation (Asn-X-Ser/Thr), 1 of which is shared with the eutherian mammals. There are over 90 potential sites for O-linked glycosylation.

Several regions of the possum ZP2 protein with potential as a reproductive vaccine have been identified using ANGIS. These peptides, their location in the ZP2 protein and the number of amino acids in common with the same region in eutherian ZP2 are shown in Table 6.

Table 6: Antigenic regions in the Brushtail possum ZP2 protein that may be suitable for use as contraceptive vaccine.

Possum ZP2 Peptide Sequence Amino Identity to Acid Eutherian No. ZP TSIALQVGSILIAEIFFVAVLCLVKCM 679-705 QRLTLSSRMVCILGPVTCN 249-267 47% IFRCAHVVTSENLILRATYKSCAERVHGTYRVNLKFL 83-119 PQVAWTVIVGYS 178-189 33% LYTVALQLTYG 235-245 NVPVNLS 291-297 29% S: TSGVAVE 299-305 29% It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

SEQUENCE LISTING <110> Marsupial CRC Limited <120> Marsupial contraceptive vaccine <130> 91189 <150> <151> <150> <151> P08354 1997-07-31 P018 00 1998-02-12 <160> a a.

a <170> Patentln Ver. SEQ ID NO: 1 <211> 2182 <212> DNA <213> Trichosurus vuipecula <400> 1 aaaatgtgga gaaggctctt tggagtcagg ctaactgggg cCcccaaagg gacatgccct agcccacagc ttccctggct cttctgcctt gtggccctgg taacctctgc agtggttggt 120 aacttctcaa ggatttcctg aatgaaatct tataagagtt aatgagacta gatgaggttc tcattttcgc gcttggactg atgcagcagg aatgctgctg cagcttacat ttaggtccag gcattgacag ggcgttgctg tctaaaatgt tttcagtatt gtttcagtag agccaccaaa cagccggcct tgtggaacaa ctttgggcag agatgctatt ccaattgtgt 25 tcctatctgc atttacatgg gactgttggg gatggctgtg gtgaactatc ggccaagctc catccagatt ataattqaag caagccccct attgcccttc ctaccagctt aagatcttgg ttcgttgcgc gtgctgaaag cgagcaacca taggtgaaat agatcctacc tcattgtggg gctacagttt gagtcttgca atggacctcc tcacctgtaa ccataagcat tagagtcgag ctgagagtgg atggggaggt ttgcaggtgg cgaagcctgc ttaagaatcc gagcaaagtt acctcccccc atagtggcac cagtgaaacc agccttatag aagtacaagt caacactgtc attatacact ccaatcacta tctctagcct ctcctttgtgI aaaagtctac catttagaggz aagtagggtct cccaggcaca caacaagtct ccatgttgtg ggtgcatggt ggttgtaacc gttagcagcc cagtt t tgat tgatagcccc tgtcattgag ctatgagcaa tgaacagagg ttcaacacac agagaatcgt aaatggctca tgcaggtgtt ggtatctgtg cacatgcacc cttgaacctg ctctcaggac tgaaggaggc aaacaaaatt tgacctcatg aggaccactt ggatgatcag agtaaataga tgtggaccct ggacaactat ccaaaggttt gatttatttc ttccgtgact iatagcaagt ~cgaacaaac :atcctaata gtcacttgct tggcaagcat acctcagaga acgtatcgag taccaggtca acgaactgta gatgagacta agaatgcaaa aacagtaaaa gaaaacaatc cttaccctct atgactctta aacgttcccg agactacatt cagttttact atcatctatc tccgatggct gatacactcc: atggttcgat caagccatct actagagaca gtcaggacaa tccttagtcc tacccaatag aatgacccaa atgtctcttc cgcacaaaat gaagtgacaa cactgcagtg tgtcctgggt cttcctggac actgaagggc- 9cagagatat atgatgatga atgtggttga acctcatctt tgaatctcaa gctgtcctgc ccaaggattt tgggtagaga cccttacact tcatcttgcg acctgtatac cgtcccgaat ttataccaga tgaatttgtc tcaacaaaag tgccttcact ctgaattctg ttatggactt aagtaagaga ttcatatacc atgaaaatga gtgaattcag atatcagcag ttcagatcta tgagatacct acatcaaact cccgatggaa tccaccatgt cttttgcctt tcttgctctg catctaggac ctgtcttctt cttggtatga tttttgtggc *aatgaagatt 180 ttcctttggc 240 gagagctacg 300 attcctgccc 360 cattcaagct 420 catgtctgtt 480 acctcaggtg 540 tcaggaagcc 600 agtgtccttc 660 tgtggctctc 720 ggtttgcata 780 gtttccaggg 840 aaagaccagt 900 aattctgaaa 960 aaagctgaga 1020 tgagtccccg 1080 tgaagtctac 1140 tacagcctgc 1200 cctgaatggg 1260 aatccatgct 1320 tctgacagtg 1380 tcctcctcct 1440 cccagacgag 1500 gcgtcaacca 1560 cgtcttagat 1620 tgtcattgtg 1680 aggctcttca 1740 tgtatctggt 1800 tgaccaattc 1860 aaaaagagat 1920 ggtatcagat 1980 aggaacaagt 2040 agtgctttgt 2100 4 4 ctggttaaat gcatgccagg caggacaaga gctgtgagtt aacaaatgag ctgcttaata 2160 aaatcatcca aacatcaaaa aa 2182 SEQ ID NO: 2 <211> 712 <212> PRT <213> Trichosurus vuipecula <400> 2 met Trp Arg Arg Leu Phe Gly Val Arg Leu Thr Gly Ala Pro Lys Gly 1 5 10 3 9 U 9 9 U ,.o

S

U

*9 *9 9 Thr Val Thr Leu 65 20 Giu Arg Val Thr Glu 145 Phe Pro Thr Pro Gin Val Val Tyr Asp Ser Trp 70 Cys Ala Lys Ser Phe Leu Ser Cys Ala Thr 150 Leu Pro 165 Trp Thr Gin Glu T rp Phe Met T yr Val Gi u 105 Giu Ile Thr Asp Val 185 Gin Phe Thr Ile Vai S er Vai Thr Al a Asp 155 Glu Asp Gi y 195 200 205 Giu Asn Ser Lys Ile Ile Leu Arg Val Ser Phe Asn Ala Ala Gly Val Let 22! Let Val IlE Arg Ser 305 Lys Lys Pro Thr Pro 385 Pro 25 Leu Tyr Ile Gi y 4 65 Ile 35 Pr o 210 i His Tyr iThr Tyr Cys Ile Ile Pro 275 Asn Vai 290 Arg Asn Met Ser Leu Arg Glu Phe 355 Ser Asp 370 Ala Leu Ala Phe Asn Gly Giu Asn 435 Thr Arg 450 Thr. Asp Val Ser Asp Giu Giu Gi y Leu 260 Giu Pro Gi y Giu Phe 340 C ys Gi y Asn Lys C ys 420 Glu Asp Leu Val1 Ser *Gin Pro 245 Gi y Phe Val S er Ser 325 Gin Giu Phe Leu Asn 405 Gly Ile Ser Met Lys 485 Tyr Cit.

230 Prc P rc Pro Asn Arg 310 Giy Tyr Ser Met Asp 390 Pro Thr His Glu iai 470 P ro ,eu 215 1Asn Giu Val Giy Leu 295 Leu Al a Tyr Pro Asp 375 Thr Ser Arg Al a Phe 455 Arg Gi y Gin *Asn His Leu Gin Arg Leu 250 Thr Cys Asn 265 Ala Leu Thr 280 Ser Lys Thr His Phe Asn Gly Val Gin 330 Gly Glu Val 345 Val Ser Val 360 Phe Glu Vai Leu Gln Val Gin Asp Met 410 Ala Lys Phe 425 Leu Trp Ala 440 Ser Leu Thr Thr Asn Ile Pro Leu Ser 490 Pro Tyr Arg 220 Thr Leu 255 Ser Ala S er Lys 31i5 Phe Val Val Tyr Arg 395 Val Giu Asp Vai Ser 475 Leu Asp *Thr Ile Gl y 300 Ar g Tyr Ser Al a Ser 380 Asp Arg Gi y Leu Arg 460 Ser Vai Asp His Set 285 Val Ile Leu Val1 Gi y 365 His Thr Phe Gi y Pro 445 Cys Pro Leu Gn Met 270 Ile Al a Leu Pro Ile 350 Gi y Gin Al a His Gin 430 Pro Tyr Pro Gin Tyr Thr Giu Val Lys Ser 335 Ile Thr Thr Cys Ile 415 Al a As n Tyr Pro Ile 495 Pro *Leu Asn Giu Ser 320 Leu T yr C ys Lys Gin 400 Pro Ile Lys Ser Pro 480 Tyr Ile

S

S. S 500 Val Arg Tyr Leu 515 Arg Gin Pro Ile Tyr Met Gi u Val Arg Leu 545 Gi y Gly Thr Phe Leu 625 Ile Val, Pro Ile As r 530 Ser C ys Ser Phe His 610 Cys Gi u Ser Trp '1 a Asp Val Asp Ser Al a 595 Cys Ser Giu Asp Tyr 675 GiuI Pro Asp Tyr Val 580 Phe Ser Val Lys Gin 6 60 ilu lie Asn Pro Thr 565 Asn Val Vai Thr Ser 645 Aila Giy Phe Ile Lys 535 Met Ser 550 Leu Asp Tyr Pro Ser Gly Leu Leu 615 Cys Pro 630 Thr Ile Pro Ser P Thr Ser I Phe Val A 69 5 520 Leu Val Leu Asp Asp 540 Leu Pro Arg Trp Asn 555 Asn Tyr Arg Thr Lys 570 Asn His Tyr Gin Arg 585 Gly Gin Ala Leu Ser 600 Cys Asp Gin Phe His 620 ily Ser Ser Arg Thr 635 ki a Ser Leu Pro Gly 650 'he Arg Giy Arg Thr 665 :ie Ala Leu Gin Val Gir 525 C ys Val Phe Phe Ser 605 Pro Lys Pro Asn Ci y 510 1Val Trp Ile His Gi u 590 Leu Asp Arg Val ThrC 670 Ser I Val Al a Val His 575 Vali Ile Ser Asp The 655 3U lie *Asn *Thr Asp 560 Val Thr Tyr Pro Ile 640 Leu Gi y Leu

S

80 la. Val 685 Leu Cys Leu Val Lys Cys Met 700 690 Gly Arg Thr Arg Ala Val Ser 710 SEQ ID NO: 3 <21i> 1270 <212> DNA <213> Macropus eugenii <400> 3 tggggactgg gctggaacca tccattctcc ccaccccctg tgtaaacagg gatttctttg tccttcggcc tgtgccccaa gggactccat gagtgtggaa tgtgctccat tatacaccaa ctcagtcccc atccggtgtc tcctacctgg gtccctttcc acgcctcatg gcagatgact cttgatccac attcaggctg tgatcgctgc acagccacct tgacttcaat gggtgcctgg tcctcggccc gggcagaatg ctctaggaat gagatctata cagccccttg aacaaagcct aggccctagt gacatctgta caggaaacgg agcctagcaa gatgttgggg cctctggtcc caatgtagga gacatcccag ggctacagtc tgcctggtgc ctccaggaat gtctagggat aaaaaaaaaa aggtcctctc tggtagacgt gaactgggca tgcccgcaga gtgaactcca.

acctctcgca aataccccag attccaccct ggagtacaga aggtctacag tgaccccaga.

tagatggaca tactgcgttt tcacctgcca gttcctataa cctgctgcaa.

atcaggaacc tatctgaggc aatggccaga tgtgcctaat tctaccactg ctgggttacc gcaatgtgag gctggtccag ccccctgaat gatgactcca.

gagcccagtg gagagacaat gtccagggag gaggatctct tggttaccat cccagtctct gtcacatgac catggtagac cctaaaagtc cttaacaact gacaagaacc aggtaaccct tgagaatggg actgctgctg cgtaggcagc aaataaagtg cagcaccctg gatgaccggc gctgcagagc aagagagtca gactcattca gtccttagaa gtgagcagca cagcggctca tcagccttcc gtgcccctga gtcccctacc tcttcctcaa tccttcaagt acaaccactg gacatgtggg tgcacacacc tcagaattag ccaaaactgg ttgttgatgg cacaaataca aaggaaaaca ctctgggatc tggtagtgag tgaccctggg tctttgaggt tctacagtac gcagtcctgt gagccatcct agttttccct agctagggga.

ggctctttgt atgttatcat tcttcatctc ttgcccaaga accaggtccc tccctgtaga tctcatcctc aggctgatct gggaagagaa taggggtagc gatttccttg caaaaaaaaa 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1270 SEQ ID NO: 4 <211> 404 <212> PRT <213> Macropus eugenji <400> 4 Gly Asp Trp Ala Gly Thr Lys Val Leu Ser Trp Val Thr Gin His Pro 1 510 Ala Leu Gly Ser Pro Phe Ser Pro Pro Pro Val Val Asp Val Gin Cys 20 25 Glu Asp Asp Arg Leu Val Val Ser Val Asn Arg Asp Phe Phe Gly Thr 40 Gly Gin Leu Val Gin Ala Ala Glu Leu Thr Leu Gly Pro Ser Ala Cys 55 Ala Pro Met Pro Ala Asp Pro Leu Asn Lys Arg Val Ile Phe Glu Val 65 70 75 Gly Leu His Glu Cys Gly Ser Glu Leu Gin Met Thr Pro Asp Ser Phe 90 Ile Tyr Ser Thr Val Leu His Tyr Thr Pro Asn Leu Ser Gin Ser Pro 100 105 110 Val Val Leu Arg Ser Ser Pro Val Ser Val Pro Ile Arg Cys Gin Tyr 115 120 125 Pro Arg Arg Asp Asn Val Ser Ser Arg Ala Ile Leu Pro Thr Trp Val 130 135 140 Pro Phe His Ser Thr Leu Ser Arg Glu Gin Arg Leu Lys Phe Ser Leu 145 150 155 160 Arg Leu Met Ala Asp Asp Trp Ser Thr Glu Arg Ile Ser Ser Ala Phe 165 170 175 Gin Leu Gly Asp Leu Ile His Ile Gin Ala Glu Val Tyr Ser Gly Tyr 180 185 190 His Val Pro Leu Arg Leu Phe Val Asp Arg Cys Thr Ala Thr Leu Thr 195 200 205 Pro Asp Pro Val Ser Val Pro Tyr His Val Ile Ile Asp Phe Asn Gly 210 215 220 Cys Leu Val Asp Gly Gin Ser His Asp Ser Ser Ser Ile Phe Ile Ser 25 225 230 235 240 Pro Arg Pro Gly Gin Asn Val Leu Arg Phe Met Val Asp Ser Phe Lys 245 250 255 Phe Ala Gln Asp Ser Arg Asn Glu Ile Tyr Ile Thr Cys His Leu Lys ':260 265 270 Val Thr Thr Thr Asp Gin Val Pro Ser Pro Leu Asn Lys Ala Cys Ser 275 280 285 Tyr Asn Leu Thr Thr Asp Met Trp Val Pro Val Glu Gly Pro Ser Asp 290 295 300 Ile Cys Thr Cys Cys Lys Thr Arg Thr Cys Thr His Leu Ser Ser Ser 35 305 310 315 320 Arg Lys Arg Ser Leu Ala Asn Gin Glu Pro Gly Asn Pro Ser Glu Leu Glu Ala Asp Gly Pro Lys 355 Pro Glu Leu 370 Met Leu Gly Pro Leu 345 Asn Asn 330 Val Val 335 Leu Gly Giu Glu Leu Leu Leu Leu 375 Cys Leu Ile Val.

390 Leu Ser Glu Ala Glu Asn 350 Gly Asp Ile Pro Giu Trp 365 Val Ala Ala Thr Val Cys Val Gly Leu 385 ser Val Leu Gly Ser His Arg Phe Pro Arg Asn Val SEQ ID NO: <211> 1326 <212> DNA <213> Trichosurus vuipecula <400> 4 4* *4 4 4* S a 4*

S**

a a

S

4 a

S

4* 54 atggagctgg tgggctacaa tctccacccc agggact tct gcttgtgccc 25 catgagtgtg cattatgcac cccatccagt tgggtccctt atggcagatg cacatccagg tgcacagcca aatgggtgcc cctgggcaga aatgagatct 35 ttgaacaaag agggacatct gagctaggct gcaaggccct ctgtggtggc ttggaactgg ctgtacctgc ggagtgaact caaatctctc gtcaataccc tccattctac actggagtac ctgatgtcta ccttgacccc tggtagatgg atgtgctgcg atatcacctg cctgttccta gcagctgctg agtgtctgtt ctcctgggct agtgcagtgt acggctggtc agagcccctg ccagatgacc ccagagtcca caggagagac cctgtccagg agagaggaca cactggctac agacccagc acagtcacgt tttcttggta ccacctaaaa taactcaaca ccagacagga cttttactct acccagagcc gaggatgacc caggctgcag agtaagagag ccagactcgc ctggtcctta aatgtgagca gagcagcggc tcttcagcct catgtgcctc tctgtcccct gactcttcct gactccttca gtcacagcca gctgacgagt acctgcatat gggctctcca ctgctccagg ggctagtagt agctgaccct tcgtctttga tcatctacag gaagcagtcc gcagaggcat tcaagttttc tccagctggg tgaggctctt accatgttgt caatcttcat ggtttgccca ctgaccaggc ggctccctgt ccctctcatc ggatggggac atcttcattc 120 gagtgtgaac 180 gggcccttcg 240 ggtgggactc 300 cactgtgctc 360 agtatcagtc 420 ccagcccacc 480 cctacgcctc 540 ggatttgatc 600 tgttgatagc 660 cattgacttc 7290 ttctccccgg 780 agactctagg 840 ccccagtccc 900 ggaaggccct 960 ctccaggaaa 1020 cggagcctag cagatcagga accaggtaac: ccttcagaat gggcctctgg ttctatctga ggctgagaat gggccaaaat ggaggcatcc caaaatggcc: ggagctgctg ctggggttga gtctgcctac tgctgggcct catcataggc agccacaaat aatgtctagg gcttctatca ctctgaaata aagtgaagga aaaaaa ttgaggctga cctgatgctg 1080 cggggcaagg gaacaatgtg 1140 ctgtagggat agcggctaca 1200 tcgggtctcc ttgctccagg 1260 aaaccaaaaa aaaaaaaaaa 1320 1326 SEQ ID NO: 6 <211> 422 <212> PP.T <213> Trichosurus vulpecula <400> 6 Met Glu Leu Gly Ala Arg Leu Val Ser Val Leu Leu Leu Trp Ala Leu 1 Gin Asp Gly Ser Pro Ala Trp Ala Thr Ser Leu Ser Trp Ala Thr Gin Val Ala Val Asp Phe Phe Gly Ser Ser Pro Pro Pro Gin Cys Asp Asp

C

4

C.

C

C.

C

C. C Arg Leu Val Gin Val Ser Val Giy Thr Gly Arg Leu 65 25 Ala Cys Ala Pro Val 85 Giu Val Gly Leu His 100 Ser Leu Ile Tyr Ser 115 Ala Ala Giu Leu Gly Pro Pro Ala Glu Pro Ser Lys Arg Val Val Phe Giu Cys Gly Thr Val Leu 1-20 Arg Ser Ser Leu Gin Met Tyr Ala Pro Thr Pro Asp 110 Leu Ser Gin Ile Gin Cys Ser Pro 130 Gin Tyr 145 35 Trp Val C C. C. C C

C.

Leu Val Leu Pro Arg Arg Pro Phe His 165 13s Asp Asn 150 Ser Thr Val Ser Ser Arg 155 Leu Ser Arg Glu 170 Ile Gin Pro Pro Val Ser Val Gin Arg Leu Lys 175 31 Ser Leu Arg Leu Met Ala Asp Asp Trp Ser Thr Giu Arg Thr Ser Ser 180 185 190 Ala Phe Gin Leu Gly Asp Leu Ile His Ile Gin Ala Asp Val Tyr Thr 195 200 205 Gly Tyr His Val Pro Leu Arg Leu Phe Val Asp Ser Cys Thr Ala Thr 210 215 220 Leu Thr Pro Asp Pro Ala Ser Val Pro Tyr His Val Val Ile Asp Phe 225 230 235 240 Asn Giy Cys Leu Val Asp Gly Gin Ser Arg Asp Ser Ser Ser Ile Phe 245 250 255 Ile Ser Pro Arg Pro Gly Gin Asn Vai Leu Arg Phe Leu Val Asp Ser 260 265 270 Phe Arg Phe Ala Gin Asp Ser Arg Asn Giu Ile Tyr Ile Thr Cys His 275 280 285 Leu Lys Val Thr Ala Thr Asp Gin Ala Pro Ser Pro Leu Asn Lys Ala 290 295 300 Cys Ser Tyr Asn Ser Thr Ala Asp Giu Trp Leu Pro Val Giu Gly Pro 305 310 315 320 Arg Asp Ile Cys Ser Cys Cys Gin Thr Gly Thr Cys Ile Ser Leu Ser 325 330 335 Ser Ser Arg Lys Arg Ser Leu Ala Asp Gin Glu Pro Gly Asn Pro Ser 6:09340 345 350 Giu Phe Giu Ala Asp Leu Met Leu Gly Pro Leu Val Leu Ser Giu Ala 00355 360 365 Giu Asn Gly Pro Lys Ser Gly Gin Gly Asn As9n Val Gly Gly Ile Pro 370 375 380 Lys Trp Pro Giu Leu Leu Leu Gly Leu Thr Val Gly Ile Ala Ala Thr e *385 390 395 400 *0*Val Cys Leu Leu Leu Gly Leu Ile Ile Gly Ser His Lys Phe Gly Ser 405 410 415 Pro Cys Ser Arg Asn Val too* SEQ ID NO: 7 00 0 .00. <211> 27 <212> PRT <213> Trichosurus vuipecula <400> 7 Thr Ser Ile Ala Leu Gin Val Gly Ser Ile Leu Ile Ala Giu Ile Phe 1 5 10 Phe Val Ala Val Leu Cys Leu Val Lys Cys Met SEQ ID NO: 8 <211> 19 <212> PRT <213> Trichosurus vuipecuia <400> 8 Gin Arg Leu Thr Leu Ser Ser Arg Met Val Cys Ile Leu Gly Pro Val i 5 10 Thr Cys Asn SEQ ID NO: 9 <21.1> 37

PRT

<213> Trichosurus vuipecula <400> 9 *Ile Phe Arg Cys Aia His Val Vai Thr Ser Giu Asn Leu Ile Leu Arg 1 5 10 .*Ala Thr Tyr Lys Ser Cys Ala Glu Arg Val His Gly Thr Tyr Arg Val 20 25 Asn Leu Lys Phe Leu SEQ ID NO: <211> 12 <212> PRT <213> Trichosuru5 vuipecula <400> Pro Gin Val Ala Trp Thr Val Ile Val Gly Tyr Ser SEQ ID NO: 11 <211> 11 <212> PPRT <213> Trichosurus vuipecula <400> 11 Leu Tyr Thr Val Ala Leu Gin Leu Thr Tyr Gly 1 5 SEQ ID NO: 12 20 <211> 7 <212> PRT <213> Trichosurus vuipecula <400> 12 25 Asn Val Pro Vai Asn Leu Ser a a..

*aa.

*a.

SEQ ID NO: 13 <211> 7 <212> PRT <213> Trichosurus vulpecula <400> 13 Thr Ser Gly Val Ala Val Glu 1 SEQ ID NO: 14 <211> <212> PRT <213> Trichosurus vulpecula <400> 14 Ser Leu Ser Ser Ser Arg Lys Arg Ser Leu Ala Asp Gin Glu Pro 1 5 10 SEQ ID NO: <211> <212> PRT <213> Macropus eugenji <400> His Leu Ser Ser Ser Arg Lys Arg Ser Leu Ala Asn Gin Giu Pro 1 5 10

REFERENCES

Castle, PE, Dean, J (1996) Molecular genetics of the zona pellucida: implications for immunocontraceptive strategies. In SK Gupta, C Doberska (eds): "Zona Pellucida Glycoproteins and Immunocontraception". J Reprod Fertil Suppl 50: 1-8.

Caughley Shepherd and Short J. (1987) 'Kangaroos, their Ecology and Management in Sheep Rangelands of Australia' (Cambridge University Press: Cambridge) Chang, YS, Hsu, CC, Wang, SC, Tsao, CC, Huang, FL (1997) Molecular cloning, structural analysis, and expression of carp ZP2 gene. Molec. Reprod.

Dev. 46: 258-267.

Cowan, P.E. (1990). Family Phalangeridae. In'The Handbook of New Zealand Mammals'. (Ed. C.M. King) pp. 67-98. (Oxford University Press: Auckland).

Epifano, O. Dean, J. (1994). Biology and structure of the zona pellucida: a target for immunocontraception. Reprod Fertil Dev 6, 319-30.

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Claims (32)

1. An isolated polynucleotide, the polynucleotide including a sequence characterised by nucleotides 4 to 2142 of SEQ ID NO:1; (ii) a sequence characterised by nucleotides 1 to 1216 of SEQ ID NO:3; (iii) a sequence characterised by nucleotides 1 to 1269 of SEQ ID NO:5; or (iv) a sequence which hybridises selectively under stringent conditions to a sequence as defined in any one of paragraphs to (iii) above.

2. A polynucleotide sequence according to claim 1 wherein the polynucleotide is less than 5000 nucleotides in length

3. A polynucleotide sequence according to claim 2 wherein the polynucleotide is less than 1000 nucleotides in length.

4. A polynucleotide sequence according to claim 3 wherein the polynucleotide is less than 500 nucleotides in length.

5. A polynucleotide sequence according to any one of claims 1 to 4 20 wherein the polynucleotide is at least 18 nucleotides in length. An isolated polynucleotide having a sequence selected from: a sequence characterised by nucleotides 4 to 2142 of SEQ ID NO:1; (ii) a sequence characterised by nucleotides 1 to 1216 of SEQ o. 25 ID NO:3; (iii) a sequence characterised by nucleotides 1 to 1269 of SEQ ID NO:5; or (iv) an allelic variant or functional equivalent of a sequence as defined in any one of paragraphs to (iii) above.

7. An isolated polynucleotide according to claim 6 wherein the allelic variant or functional equivalent shares at least 70% homology with a sequence shown in any one of SEQ ID NOS: 1, 3 or 5, wherein the homology is calculated by the BLAST program blastn as herein described.

8. An isolated polynucleotide according to claim 7 wherein the allelic variant or functional equivalent shares at least 90% homology with a sequence shown in any one of SEQ ID NOS: 1, 3 or 5, wherein the homology is calculated by the BLAST program blastn as herein described.

9. A vector including a polynucleotide according to any one of claims 1 to 8.

10. A vector according to claim 9 wherein the vector is a bacterial vector, a baculovirus expression vector or a Semliki Forest Virus expression vector.

11. A bacterial, yeast, insect, plant or mammalian host cell transformed with a vector according to claim 9 or claim

12. A method of producing a marsupial ZP2 or ZP3 polypeptide which includes culturing a host cell according to claim 11 under conditions enabling the expression of the polypeptide and optionally recovering the polypeptide.

13. A method according to claim 12 wherein the conditions enable glycosylation of the polypeptide.

14. A marsupial ZP2 or ZP3 polypeptide produced by a method according to claim 12 or claim 13.

15. A marsupial ZP2 or ZP3 polypeptide according to claim 14 wherein the ZP2 or ZP3 polypeptide is glycosylated.

16. An oligonucleotide probe or primer of at least 8 nucleotides, the 20 oligonucleotide having a sequence that hybridises selectively under stringent conditions to a polynucleotide according to claim 1.

17. An oligonucleotide probe or primer according to claim 16 in which the oligonucleotide comprises at least 18 nucleotides.

18. An oligonucleotide probe or primer as claimed in claim 17 in which 25 the oligonucleotide comprises at least 25 nucleotides.

19. An oligonucleotide probe as claimed in any one of claims 16 to 18 in which the oligonucleotide is conjugated with a label.

20. An oligonucleotide probe according to claim 19 in which the label is i selected from the group consisting of a radioisotope, an enzyme, biotin, a fluorescent molecule or a chemiluminescent molecule.

21. An isolated polypeptide having a sequence which is: as shown in SEQ ID NO: 2; (ii) as shown in SEQ ID NO: 4; (iii) as shown in SEQ ID NO: 6; or (iv) a derivative or biologically active fragment of a sequence as Sdefined in any one of paragraphs to (iii) above.

22. An isolated polypeptide according to claim 21 wherein the derivative shares at least 50% homology with a sequence shown in any one of SEQ ID NOS: 2, 4 or 6, wherein the homology is calculated by the BLAST program blastx as herein described.

23. An isolated polypeptide according to claim 22 wherein the derivative shares at least 70% homology with a sequence shown in any one of SEQ ID NOS: 2, 4 or 6, wherein the homology is calculated by the BLAST program blastx as herein described.

24. An isolated polypeptide according to claim 21 wherein the biologically active fragment includes a sequence selected from the group consisting of: TSIALQVGSILIAEIFFVAVLCLVKCM (SEQ ID NO: 7); (ii) QRLTLSSRMVCILGPVTCN (SEQ ID NO: 8); (iii) IFRCAHVVTSENLILRATYKSCAERVHGTYRVNLKFL (SEQ ID NO: 9); (iv) PQVAWTVIVGYS (SEQ ID NO: LYTVALQLTYG (SEQ ID NO: 11); (vi) NVPVNLS (SEQ ID NO: 12); and (vii) TSGVAVE (SEQ ID NO: 13). 20 25. An isolated polypeptide according to claim 21 wherein the biologically active fragment includes a sequence selected from the group consisting of SLSSSRKRSLADQEP (SEQ ID NO: 14) and HLSSSRKRSLANQEP(SEQ ID NO:

26. A chimeric polypeptide comprising a first polypeptide, the first polypeptide being a polypeptide according to any one of claims 21 to 25, and a second polypeptide.

27. A chimeric polypeptide according to claim 26 wherein the second polypeptide is selected from keyhole limpet hemacyanin (KLH) and tetanus toxoid (TT).

28. A composition for use in raising an immune response in animals against the ZP2 or ZP3 protein, the composition including an acceptable carrier and a polypeptide according to any one of claims 21 to 27.

29. A composition for use in raising an immune response in animals against marsupial ZP2 or ZP3 proteins, the composition including a polynucleotide according to any one of claims 1 to 8. 41 A composition according to claim 29 wherein the polynucleotide is incorporated into a multiclellular organism.

31. A composition according to claim 30 wherein the multiclellular organism is a parasitic nematode.

32. A composition according to claim 29 or claim 30 wherein the composition is in the form of a live vaccine.

33. A composition according to claim 32 wherein the live vaccine is a Salmonella vaccine or replication limited Macropod Herpesvirus vaccine.

34. A composition according to claim 29 or claim 30 wherein the composition is in the form of a bacterial ghost vaccine. An antibody which binds selectively to a polypeptide according to any one of claims 21 to

36. A method of inhibiting fertilization of an egg by a sperm in a female marsupial which method includes inoculating the female marsupial with a polypeptide according to any one of claims 21 to 27. 36. A method according to claim 36 wherein the marsupial is a possum or macropod or other species as listed in Table 1 herein. SDated this tenth day of April 2001 Marsupial CRC Limited Patent Attorneys for the Applicant: F B RICE CO