WO1998000536A1

WO1998000536A1 - Immunocontraceptive methods and peptide or polypeptides for use in these methods

Info

Publication number: WO1998000536A1
Application number: PCT/GB1997/001740
Authority: WO
Inventors: Michael John Taussig; Derek Bryan Alfred Symons
Original assignee: The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
Priority date: 1996-06-29
Filing date: 1997-06-26
Publication date: 1998-01-08
Also published as: GB9613705D0; AU3350097A; CA2259237A1

Abstract

The production of anti-sera to small peptides or polypeptides which cross react with uteroglobin is disclosed giving rise to diagnostic and detection as well as immunocontraceptive methods. In particular, a method for controlling the fertility of a female mammal, especially a feral mammal such as wild rabbits, is described. This method comprises administering to a mammal a peptide or polypeptide which stimulates an immune response, said response including production of elements which bind uteroglobin and reduce fertility of said female mammal. Peptides or polypeptides which may be used in the method include peptides or polypeptides comprising (a) uteroglobin or a fragment thereof, a peptide or polypeptide derived from uteroglobin, or a variant or peptide mimetic of any of these, and (b) a carrier protein. This may be applied in a conventional vaccine formulation, including using viral vaccine vectors. Preferably, they are formulated into compositions which are suitable for oral administration. Novel peptide or polypeptides for use in the method, their production and other aspects are also described and claimed.

Description

Immunocontraceptive Methods and Peptide or Polypeptides for Use in these

Methods

The present invention relates to contraceptive methods effective in mammals and particularly rabbits, to peptides and polypeptides which are useful in those methods, to processes for their production and to pharmaceutical compositions containing them.

The development of a contraceptive vaccine, particularly for wild or feral species of mammals such as wild rabbits, would be desirable in order to achieve a level of control over the population levels of these mammals.

Uteroglobin (UG) is the predominant protein in the uterine lumen of rabbits during the pre- implantation phase of pregnancy, accounting for up to 40-50% of the protein content of the uterine secretion in early pregnancy. It is a secreted protein of MW 14kD comprising a dimer of two identical chains of 70 amino acids, linked by disulphide bridges. The structure has been fully characterised (Morize I et al., J. Mol. Biol. (1987) 194: 725-739) and comprises three helical regions interconnected by interhelical loops (Figure 1).

Several properties of UG have been identified, including binding of progesterone, inhibition of inflammation and immunosuppressive effects by masking the antigenicity of sperm and embryos within the female tract.

The high incidence of UG in the pregnant rabbit uterus implies an important role for this protein in pregnancy. Antisera to UG have been raised in various species such as sheep and mice. It has been shown that anti- rabbit UG antibodies raised in chickens and administered passively to rabbits 2-6 days post-coitum prevented implantation (Krishnan R.S. Experientia 27: 955-956).

The applicants have found that under certain circumstances, an immune response can be stimulated in mammals such as rabbits, against uteroglobin or to polypeptides or small peptides based upon uteroglobin. Furthermore, it has been found that the response can inhibit fertility of the mammal so giving rise to an immunocontraceptive.

The present invention provides a method for controlling the fertility of a female mammal, said method comprising administering to said mammal an agent which stimulates an immune response, wherein the response includes the production of an element which interacts with uteroglobin of said mammal so as to reduce the fertility thereof.

Suitable agents for use in the method comprises a peptide or polypeptide which stimulates an immune response, said response including production of a binding element which specifically binds uteroglobin so as to reduce fertility of said female mammal or an expression vector which encodes a such a peptide or polypeptide and expresses said peptide or polypeptide in vivo in said mammal.

As used herein the term 'polypeptide' is intended to encompass proteins.

Binding elements will generally comprise immunoglobulins and in particular antibodies. In order to achieve a contraceptive effect, it may be necessary to deliver the peptide or polypeptide using an appropriate delivery strategy as is conventional in the art.

Preferably, the method will comprise administration of a peptide or polypeptide which stimulates an immune response, said response including production of a binding element which bind uteroglobin and reduce fertility of said female mammal.

Novel peptides or polypeptides for use in this method, which are able to stimulate an immune response in a mammal, said response having an effect on uteroglobin so as to reduce the fertility of a female mammal, form a further aspect of the invention.

Suitably, the peptide or polypeptide comprises uteroglobin or a fragment thereof, or a variant or peptide mimetic of any of these, which is coupled to a carrier protein. Preferably, the peptide or polypeptide is an autologous peptide or polypeptide which is native to the target mammal Thus for example, where the target mammal is a rabbit, the polypeptide used is preferably rabbit uteroglobin or a fragment thereof, which has been rendered immunogenic for example by coupling to a carrier

Peptides or polypeptides which produce an immune response, such as an antibody response, which cross-reacts with uteroglobin may also be useful for diagnostic purposes Antibodies raised against such peptides or polypeptides may be used to detect the presence of uteroglobin in samples Alternatively, the antibodies may be used in passive immunisation methods Techniques for using such antibodies in detection and diagnosis are well known They may include ELISA techniques, or the use of labelled antibodies or anti-antibodies, for example gold labelled antibodies Both competitive and direct assays may be formulated in a conventional manner

Thus the invention also provides a peptide or polypeptide which comprises uteroglobin fragment, a peptide or polypeptide derived from uteroglobin, or a variant or peptide mimetic of any of these, which is coupled to a carrier protein and which is able to produce an immune response in a mammal in which antibodies which react with uteroglobin are produced Antibodies produced in this way and their use in diagnosis and as contraceptives in their own right form further aspects of the invention

Suitable fragments of uteroglobin are those which constitute a potential epitope These may be small fragments for example of from 4 to 25, suitably from 8 to 17 amino acids in length Indeed, small fragments are preferred as they may be easier to synthesise using chemical means and therefore they may be supplied in greater quantities at reduced cost In addition, the risk of unwanted cross-species challenge associated with release of oral vaccine in the field (the strategy anticipated for the control of feral species) may be avoidable, since small peptides can utilise species-specific motifs in protein sequences Therefore, preferably the fragments are selected so that they are largely species specific and in particular do not cross- react with other species found in similar environments such as the hare

Several fragments may be combined in a single peptide or polypeptide which has the advantage of raising a number of antibody specificities at the same time An example of such a peptide, derived from the three interhelical regions is given hereinafter and designated peptide "L".

As used herein, the term "variant" means that the peptide or polypeptide has a sequence which is similar to that of uteroglobin or fragments thereof, but wherein one or more amino acid residues are different. The changes do not alter function of the peptide or polypeptide in terms of its ability to produce an immune response in a female mammal which affects the fertility of that mammal, although the extent of that response and the resultant affect may be at a different level. For example, peptides or polypeptides which are 60% homologous to the native sequence, suitably more than 80% homologous and preferably more than 90% homologous to the native sequence and which have similar gross biological properties would constitute "variants".

The expression "peptide mimetic" used herein refers to peptides or polypeptides which are designed such that they "mimic" the function of the native uteroglobin or fragment. It is well known that in certain cases, replacement of one amino acid with another may not have a significant effect on the activity of the peptide or polypeptide. Therefore, peptides and polypeptides may be produced which are based upon the sequences of the invention but which do not resemble the sequence of the native uteroglobin protein. However, antibodies raised against such a peptide or polypeptide may be cross-reactive with uteroglobin.

In order to generate a immune response, the peptide or polypeptide should be recognised as "foreign" by the target mammal. A native protein such as uteroglobin or fragments would not be seen as foreign by its natural host. In order to induce, an immune response, it is necessary for the peptide or polypeptide to be coupled to a carrier protein. Such molecules are well known in the art. They include purified protein derivative (PPD), keyhole limpet haemocymin (KLH), bovine serum albumin (BSA) and ovalbumin (OVA).

The nature of the carrier has been found to be an important factor in determining the level and duration of the response. In particular, the greater molecular size and immunogenicity of the carrier molecule, the better the vaccine. In this respect therefore, KLH is a preferred carrier as compared to OVA. In a preferred embodiment, the uteroglobin is rabbit uteroglobin, which has an effect of reducing rabbit fertility.

Thus a particularly preferred embodiment of the invention comprises a peptide or polypeptide which comprises rabbit uteroglobin or a fragment thereof coupled to a carrier protein.

Preferably the polypeptide comprises full-length rabbit uteroglobin coupled to a carrier protein. Alternatively, one or more fragments of rabbit uteroglobin are employed. The skilled person would be able to test using routine methods, for example as illustrated hereinafter, whether any particular fragment (or combination of fragments), variants or peptide mimetics of uteroglobin have the desired activity in the target species. In particular, a useful indicator as to potential contraceptive activity would be whether the antiserum produced as a result of innoculation with the selected peptide or polypeptide cross-reacts with uteroglobin. Selection of suitable delivery techniques and formulations to ensure optimum contraceptive effect can also be determined using conventional techniques for fertility assessment, for example as illustrated hereinafter.

It has been found that in particular, peptides derived from the third helix of rabbit uteroglobin have the desired biological activity. One such peptide comprises a nonapeptide of sequence:

M Q M K K V L D S (SEQ ID NO 1)

which has been designated for the purposes of this application peptide "F".

Other immunogenic peptides are derived from one or more of the three interhelical loops. A particular example of such a peptide, which is based upon all three loops, is a peptide of sequence:

L G T P S K E F E P D D T S L P Q (SEQ ID No 2)

which has been designated for the purposes of this application as peptide "L". Peptides or polypeptides of the invention may be produced by conventional methods For example, they may be synthesised chemically using known techniques. Automated peptide synthesisers are commonly employed. These may be particularly suitable for short peptides as they can be produced quickly and easily in high quantities.

Longer polypeptides such as the protein uteroglobin itself may be obtained by purification from natural sources. Modification of the protein thus obtained may then be effected chemically in order to obtain shorter or modified fragments.

Yet a further alternative is production using recombinant DNA technology. In these cases, nucleic acids which encode the desired peptides or polypeptides are prepared, for example by isolation and cloning from natural sources, which may include amplification, or by production ab initio using known nucleic acid synthesising techniques such as automated nucleic acid synthesisers.

The nucleic acid is then introduced into an appropriate replication vector or plasmid together with suitable control sequences, such as promoters, enhancers, selection markers etc. as is conventional in the art. The replication vector or plasmid is then introduced into a host cell which may be a eukaryotic or prokarytic cell such as E. coli.

Transformed host cells are then selected and cultured and the desired peptide or polypeptide isolated from the resultant culture.

Novel nucleic acid sequences, replication vectors or plasmids, transformed cells and processes for preparing the peptides or polypeptides form further aspects of the invention.

Using recombinant DNA techniques, it may be possible to express the desired peptide or polypeptide or protein as a fusion protein with the required carrier protein. However, in general it will be necessary to couple the peptide or polypeptide or protein obtained by these methods to the carrier protein using known conjugation methods, for example using chemical linkers such as sulphosuccinimidyl 4-(N-maleidimethyl)cyclohexane-l-carboxylate or bis- diazotised-o-toluidine.

When used as an immunocontraceptive, the peptides or polypeptides are suitably administered in the form of a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier. The carriers may be solid or liquid carriers as is conventional in the art. Liquid carriers include water, saline and aqueous alcohol.

The composition may contain additional agents such as adjuvants which potentiates the immune response. Such adjuvants include Freund's complete and incomplete adjuvant, aluminium compounds such as phosphate and hydroxide, mineral oils such as squalene or biodegradable peanut oil, or mura yl dipeptide which may be incorporated into the mineral oil.

Various methods of administration of the immunocontraceptive agent can be used, following known formulations and procedures, Dosages can be determined by the skilled person and will depend upon the nature of the target animal, the particular antigen used, the mode of application etc. For example, initial doses may be divided between several sites in the animal and the number of subsequent administrations for example by injection required varies depending upon the level of response produced by the antigen. However, in general, a dosage range of lOOμg to lmg/Kg would be acceptable. For rabbits (average weight 1.5kg), suitable dosages of UG-PPD administered by injection has been found to be 200μg/animal for the initial dose, with subsequent doses of lOOμg/animal. However, when using small peptides of the invention, dosages of the complex with PPD were suitably 500μg/animal for the initial dose, with subsequent doses of 250μg/animal.

Instead of administering the peptides or polypeptides directly, they could be produced by a live vaccine. In this case, a live vector, for example an attenuated virus, such as an attenuated vaccina virus, is transformed such that it expresses the antigenic peptide or polypeptide-carrier conjugate. However, preferably, immunocontraceptive compositions of the invention are adapted for oral administration Such compositions will suitably be in the form of biodegradable microspheres as are known in the art (see for example Challacombe S J et al (1992) Immunology 76 164- 168) or liposomes (see for example Walker R I et al , Vaccine (1994) 12 387-400) and immuno stimulatory complexes or "ISCOMS" (see for example Morein B and Akerblom L (1992) in Recombinant DNA Vaccines ed Issacson R L pp369-386 Marcel Dekker New York) Such formulations may be incorporated into bait or food made available for feral populations

The peptides or polypeptides of the invention may be administered either alone or in combination with other antigens or contraceptive reagents, for example, immunocontraceptives which target sperm anitgens

Fertility trials reported hereinafter demonstrate that UG is an appropriate target for immunocontraception and that fertility is significantly reduced as a results of UG immunisation The effect is probably a result of inhibition of implantation, consistent with a role for UG in the early phase of pregnancy

The invention will now be particularly described by way of example with reference to the accompanying drawings in which-

Figure 1 is a computer derived image of rabbit UG, showing the so-called "antiflammin" peptide segment (cross hatched) and the loop peptide segments (arrows) on which peptides of the Examples are based.

Figure 2 is a graph showing the results of an ELISA assay of antiserum to peptide L, the composite loop peptide; the upper graph shows antiserum tested against the peptide, and the lower graph shows antiserum tested against native uteroglobin,

Figure 3 is a graph showing the results of an ELISA assay of antiserum to peptide F, the antiflammin peptide, the upper graph shows antiserum tested against the peptide, and the lower graph shows antiserum tested against native uteroglobin, Figure 4 is a graph showing the results of an ELISA assay of antisera of individual loop peptides 1 , 2, and 3 tested against peptide (left hand column), and antisera tested against native uteroglobin (right hand column);

Figure 5a shows the average litter sizes in groups of rabbits immunised with a variety of antigens and an irrelevant control peptide and Figure 5b shows the incidence of resorptions recorded in the experimental groups;

Figure 6 shows the time course of primary response to a peptide of the invention incoφorated in biogradable microparticles;

Figure 7 shows the response to the same peptide formulations after a booster; and

Figure 8 shows the mean antibody responses of groups of 16 rabbits to a peptide of the invention in various formulations.

Example 1

Preparation of Rabbit UG Female rabbits were given 4 subcutaneous injections, each of 5mg progesterone in 0.5ml Arachis oil BP. Injections were on days 1, 4, 5 and 6 and animals killed by i.v. Sagatal injection on day 7. The uterus was ligated at the utero-cervical junction and the lumina washed out with 1 ml sterile saline (0.15M), the washings centrifuged and the supernatants stored at -20°C.

Uterine washings from several animals were pooled, freeze dried and redissolved in a small volume of water as a means of concentration before gel filtration on a 1.8 x90 cm Bio-Gel A1.56m column (Bio-Gel Laboratories Inc), eluting with PBS, pH 7.2 at 12 mis/hour, collecting 3 ml fractions (Figure 3a). Fractions containing UG were identified by SDS-PAGE, pooled, freeze dried, redissolved and dialyses against PBS in CelluSep T2 dialysis membrane, MW cut ofT8-10kD. The UG was further purified by FPLC gel filtration on Superdex 75 HR10/30 column The resultant UG preparation did not contain other proteins detectable by gel electrophoresis. Quantitation of UG was by measurement of absorbance at 205nm.

Example 2

Preparation of Recombinant UG7

Isolation techniques such as that described in Example 1 produced relatively small yields of protein. The vector pDS-UG7 which induces high level expression of recombinant rabbit uteroglobin in bacteria (W. Peter et al., Protein Engineering (1989) 3: 61-66) was obtained The expressed recombinant UG (rUG&) forms stable dimers and binds progesterone indistinguishably from native UG. E. coli strain W3110 was transformed with pDS-UG7 and protein expression induced with isopropylthiogalactoside (IPTG). Bacteria were centrifuged, suspended in water and extracted by ultrasonic disruption. After centrifugation at 10,000xg for 15 minutes, the soluble fraction was made to lOOmM Tris-HCl (pH 7.5), 150mM NaCl and lOmM dithioerythritol (DTE). Denatured protein was removed by centrifugation at

40,000xg for 15 minutes at 4°C. Protease inhibitors were added (PMSF, ImM; EDTA, 3mM; benzamidine, ImM; leupeptin, 5mg/ml; Aprotinin l%v/v)

The protein extract was gel filtered on a Bio-Gel Al .5m column in PBS containing 0.1% sodium azide and lOmM DTE, and fractions containing rUG7 identified by gel analysis.

These were pooled, concentrated by freeze drying and further purified by FPLC on a

Superdex 75 column (Pharmacia). The identity of rUG7 was determined by comparison with molecular weight standards and highly purified UG and confirmed by amino acid sequence analysis of the purified protein.

Example 3

Preparation of Peptide Antigens

The following peptides were prepared using conventional microchemical techniques:

1. Peptide L, a peptide based on the three inter-helical loops of UG, Leu-15 to Ser-19, Lys-26 to Thr-33, and Ser-47 to Gln-50, synthesises as a single 17-mer peptide of sequence: LGTPSKEFEPDDTSLPQ (SEQ ID NO 2)

2. Three octapeptides based on the inter-helical loops of UG, synthesised with additional terminal cysteine residues for coupling to carrier and to enable multimer formation during the coupling procedure. The sequences are:-

Loopl- LLGTPSSY (SEQ ID NO 3)

Loop 2- KEFEPDDT (SEQ ID NO 4)

Loop 3- LDSLPQTT (SEQ ID NO 5)

3. Peptide F, a nonapeptide Met-39 to Ser-47 which forms the major part of the third helix of UG and has sequence similarity to the antiflammins (L. Miele et al., Nature

(1988) 335: 726-729). The sequence of this peptide is:

MQMKKVLDS (SEQ ID NO 1)

Example 4

Conjugation of Protein or Peptides to Carriers

Peptides were conjugated to PPD using sulphosuccinimidyl 4-(N- maleimidmethyl)cyclohexane-l-carboxylate (Sulpho-SMCC, Pierce Chemical Co.) as linker.

PPD was reacted with linker at pH7.5 for 30 minutes, the pH adjusted to pH 6.0 and activated PPD separated by gel filtration from uncoupled linker. Peptides of Examples 1, 2 and 3 were coupled overnight to activated PPD under nitrogen at pH 7.0. Uncoupled peptide molecules were removed by dialysis.

Bis-diazotised-o-toluidine was reacted with UG/carrier mixtures at pH7.4 for 2 hours at 4°C. Uncoupled linker was removed by dialysis. Using a similar method, other proteins or peptides including the composite loop peptide, L and UG, was coupled to both OVA and KLH.

Example 5 Immunisation Studies

Immunisation of BCG-primed rabbits using the peptide or protein conjugates from Example 4 was carried out with a minimum of two injections. Primary injections of antigen peptide-PPD were given in incomplete Freund's adjuvant both intramuscularly and subcutaneously and subsequent booster injections were given subcutaneously with a 3 week interval between injections.

Maximal responses were generally achieved after the second injection of antigen peptide-PPD conjugate. Assays of antisera to the composite loop peptide L are shown in Figure 2, to peptide F in Figure 3 and to the individual loop peptides in Figure 4. Results are shown for assays against free peptide and UG. They show that UG peptides coupled to PPD carrier are effectively antigenic in rabbits. Cross-reactivity against UG of antisera raised against small synthetic peptides demonstrates the suitability of peptides for vaccine design. Antisera to both peptides L and F showed cross reactivity (Figures 2 and 3). Of the three peptides based on loop sequences. Loop 2 antisera were strongly cross-reactive against UG, Loop 1 weakly cross reactive and Loop 3 antisera non-cross reactive.

Example 6

Effect of Immunisation on Fertility

In a fertility trial, groups of 5 female rabbits were immunised prior to mating and their subsequent fertility assessed at autopsy on day 25. Measured against a control group which were injected with an irrelevant peptide, peptide L had no effect on pregancy in this trial.

However animals immunised with the UG-PPD conjugate or peptide F-PPD conjugate showed reduced litter sizes of 22% and 15% respectively (Figure 5a). The mean litter size per animal was 5.0 and 5.4 for these groups, compared with 6.4 for those immunised with the control peptide. It was significant that there were a high number of fetal resorptions in the animals immunised with peptide F (Figure 5b). This suggests a late effect on fetal viability resulting from immunisation. Example 7 Fertility Trials

Groups of 10 female dutch rabbits were immunised with the following treatments.

Group 1 : A control peptide with no relationship to the reproductive system, Group 2 : Peptide F coupled to KLH (F-KLH); and Group 3 : UG coupled to KLH (UG-KLH).

Primary immunisations were. with the antigens in Freund's complete adjuvant; a booster injection was given 3 weeks later in incomplete Freund's adjuvant. The responses achieved with these procedures were monitored and these showed that there was a strong antibody response against UG and weaker but significant anti-UG responses after immunisation with peptide F.

Immunised female rabbits were introduced to a buck rabbit on two consecutive days for mating. Fertility was assessed on autopsy at day 25 of pregnancy. Parameters determined were the number, weight and length of viable fetuses, number of resorbed or mummified fetuses and number of corpora lutea. The results are shown in Tables I and II.

Table I

Antigen No of fetuses Weight of fetuses Weight of uterus Corpora lutea L R L R

Control 0 0 - 3 5 Peptide 4 2 168 67 4 3

0 0 - 6 3 5

2 0 49 45 3 3

3 5 188 108 4 4

1+1R 4+1R 84 137 2 6

4 3 111 128 4 3

7* -

1 2+1M 59 71 5 5

1 2 61 49 4 3

Totals 44 720 611 69

Peptide F 4 3 190 79 3 2

3+1M 4 162 87 3 4

2 0 52 56 4 6

0 0 - 0 0

0 0 - 0 1

0 0 - 5 2 2

0 0 - 7 3 6

4* 222 nd

7* nd nd

5 2 142 79

Totals 35 768 313 43

UG 0 2 71 38 3 1

2 2 121 83 5 4

2 0 58 47 4 2

0 0 - 0 0

1+1R 0 27 29 1 4

3 4 79 99 3 4

0 0 - 7 2 2

0 0 - 9 4 4

0 0 - 10 1 0

0 2 41 49 2 2

Totals 19 397 371 48

* live births Table II

Antigen Litter size¹ ±sem Reduction Litter size² ±sem Reduction

Control 4.4+0.96 5.5+0.78 Peptide F 3.5+1.1 21% 5.8±0.95 0 UG 1.9+0.83 57% 3.2±0.83 42%

¹ in group as a whole

² in pregnant animals only

The results demonstrate a marked reduction in fertility of rabbits immunised with UG: in the control group, 8 of the 10 rabbits became pregnant and produced 44 fetuses (mean litter size of 4.4 + 0.96), whereas in does immunised against UG there were 6 pregnant animals and the number of fetuses was 19 (mean litter size of 1.9 ± 0.71). This represents a reduction of 57% in total fetus number and overall litter size in UG-immunised rabbits. Among the pregnant does, litter size was reduced from 5.5 + 0.78 in the control group to 3.2 + 0.83 in the UG immunised group, a reduction of 42%. The fact that only 19 out of 48 corpora lutea (39.6%) gave rise to implantations in the UG-immunised rabbits compared with 44 out of 69 (63.7%) in controls (Table I) indicates that the effect of UG-antibodies is to prevent implantation rather than ovulation.

In does immunised against peptide F, the number pregnant was again reduced to 6, and the total number of fetuses was 35 (mean litter size 3.5 ± 1.1), a reduction of 21%.

Statistical analysis of the results in Table II showed that the overall litter size of the UG- immunised does (1.9 ± 0.71) was significantly smaller than that of the control (4.4 ± 0.96), both by the Mann Whitney U test (p-0.068) and by the two-sample T test using the minitab program (p=0.052). Comparing only the pregnant does of these two groups, the litter size of the UG-immunised animals (3.2 + 0.83) was also significantly reduced compared with the controls (5.5 + 0.78) (p=0.66). In contrast, the litter size of the peptide F immunised group (3 5 ± 1 1 ) versus control (4 4 + 0 96) was not significantly reduced (MWU test, p=0 61 ) The number of pregnant animals between the three groups was not significantly different

This trial clearly demonstrated that immunisation against UG in particular is effective as an immunocontraceptive procedure, reducing litter size through the inhibition of implantation The reduction in overall litter size of 57% was the largest effect achieved to date and was produced in a relatively large group of animals; moreover, it was statistically significant at the 95% level

Example 8

Passive immunisation with sheep anti-UG antibodies

A serum was raised by immunisation of a sheep with UG-KLH, in Freund's adjuvant The course consisted of three multi-site injections at 4-week intervals The serum IgG antibody fraction was purified by protein G affinity chromatography (ProSep G column); normal IgG from a nonimmunised sheep was prepared in similar fashion as the control

In the fertility trial, ten does were divided into two groups The test group received 4 subcutaneous injections, each of 7.5mg sheep anti-UG IgG, 2 days before and at 4, 11, and 18 days after mating, the control group received 4 injections of normal sheep IgG to the same amount Animals were autopsied on day 25 of pregnancy

The results of this trial are given in Tables III and IV

Table III

Injected No of fetuses Weight of fetuses Weight of uterus Coφora lutea L R L R

Normal 5 5 145 97 5 5 sheep IgG 0 0 9 1 2

7 2 116 143 7 1

2+lR 2 43 87 3 2

0 0 0 14 7 1

Totals 24 304 350 34

Anti-UG 2 3 96 101 2 5 sheep IgG 0 0 1 0 0

0 0 9 0 1

3 4 129 123 3 4

0 0 7 0 0

Totals 12 225 253

Table IV

Antigen Litter size¹ ±sem Reduction Litter size² ±sem Reduction

Normal 4.8±2.1 8.0+1.2 sheep IgG

Anti-UG sheep IgG 2.4+1.5 50% 6.0+0.6 25%

¹ in group as a whole

² in pregnant animals only

The results show that there was a reduction in fertility of the test group receiving anti-UG antibodies, with a 50% reduction in total fetus number; only 2 of the 5 rabbits were pregnant and produced 12 fetuses (mean litter size 2 4 + 1.5), whereas in the control group, 3 of the 5 rabbits were pregnant and produced a total of 24 fetuses (mean litter size 4 8 + 2 1) The litter size per pregnant doe (6.0 ± 0 6 was 25% lower in the anti-UG group than in the normal IgG controls (8 0 ± 1.2)

The reduction in total fetus number and mean litter size suggested that anti-UG antibodies were indeed effective in reducing fertility

Example 9 Statistical analysis of the results of Examples 7 and 8

Since both the trials of Example 7 and Example 8 compared the effect of UG antibodies versus controls, the results can be combined as shown in Table V

Table V

Antigen Litter size ±sem Reduction Litter size ±sem Reduction

Controls 4 53±0.91 6 18±0 75

UG immunised 2 07+0.66 54% 3 87+0 79 37%

' in group as a whole

² in pregnant animals only

This shows that the effect of UG immunisation on litter size is statistically highly significant (Table V) Including both pregnant and nonpregnant animals in each group, the litter size in the UG immunised group was 2 07 ± 0.66 compared with 4.53_± 0 91 in the controls, the significances being p=0.54 by Mann Whitney U test and p=0 037 by twosample T test. Taking only the pregnant does, the litter size of UG immunised animals was 3 86 ± 0 79 while that of controls was 6.17 ± 0.75, the difference again highly significant both by Mann Whitney U test (p=0.058) and p=0 037 by twosample T test (p=0 05) The overall reduction in litter size was 54%, and the reduction in litter size of the pregnant rabbits was 37% Since the number pregnant was not statistically different between control and immunised groups, the results demonstrate that the effect of UG immunisation is to reduce mean litter size significantly

Example 10 Preparation of Biodegradable Microparticles

Conjugates as prepared in Example 4 above were incorporated into microparticles by mixing a 15mg/ml conjugate aqueous solution (2ml) with 10ml of 6%w/v solution of poly(DL lactide co-glycolide) with a lactide glycolide ratio of either 50 50 or 75 25 to produce a water-in-oil emulsion This primary emulsion was mixed with polyvinyl alcohol (PVA) stabiliser to produce a water-in-oil-in- water suspension which was stirred overnight to remove solvent Microparticles were harvested by centrifugation and resuspended in water three times

Particle size range was determined using a BCA assay followed by disruption of approximately 5mg particles in 2ml of 5% w/v sodium dodecyl sulphate in 0 1M sodium hydroxide overnight Calibration curves were constructed from a series dilution of the respective conjugate Microparticles were stored freeze dried below 5°C with dessicant

Example 11

Antibody responses to peptide L after immunisation in biodegradable microparticles In order to design a vaccine which could be used for oral delivery to rabbits in the wild, antibody responses were induced to peptide L incorporated into biodegradable polylactide- coglycolide (PLGA) microparticles. These have been shown to be effective carriers for oral immunisation in rodents UG loop peptide complexed to KLH (L-KLH) or OVA (L-OVA) was incorporated into microparticles (lactide:glycolide ration 75 25) and administered to rabbits by parenteral routes in order to assess efficacy

Following sub-cutaneous injection of L-KLH or L-OVA, antibodies were assayed over time at a serum dilution of 1.100 and the results are shown in Figure 6 Antibodies were induced quickly, peaking at 20 days, and then declining, the response to L-KLH was stronger than that to L-KLH The animals were then rechallenged and in response to L-KLH, made a strong and long- lasting secondary respone, the level of which was still high after 200 days (Figure 7). The response to L-OVA also peaked well. This shows that microparticie-incorporated antigen is an effective means of parenteral delivery, indicating that it will also be an effecive means of oral administration.

Example 12

Comparison of the efficicacy of different formulations

Using the methodology of Example 1 1, four groups of 16 rabbits each received L-KLH in a different formulation as follows:

Group 1 : L-KLH in complete Freund's adjuvant;

Group 2: L-KLH in saline;

Group 3 : L-KLH in 50:50 lactide:glycolide microparticles; and Group 4: L-KLH in 75:25 lactide:glycolide microparticles.

The microparticle formulations administered to Groups 3 and 4 provide different rates of antigen release (fast and slow respectively).

The mean antibody responses following a single injection are shown in Figure 8. The response in Group 1 was the largest and most sustained, while that of Group 2 peaked well at around 20 days but declined rapidly thereafter. Of Groups 3 and 4, Group 4 gave the superior response with a susiained peak between days 20 and 60 and declining thereafter.

Example 13

Fertility Trials with Animals following immunisation with L-KLH

The animals in the four groups described in Example 12 were boosted and entered into a fertility trial, the results of which are shown in Table VI and VII. Table VI

Antigen No of fetuses Weight of fetuses Weight of uterus Coφora lutea L R L R

Peptide L, 4 4 108 95 5 3 saline 3 5 131 101 3 6

2 2 32 64 6 5

1 3 69 55 3 3

Totals 24 340 315 34

Peptide L, 0 0 - 1 1

Freund's 4 3 94 91 6 3 adjuvant 0 0 - 8 0 0

4 3 127 104 4 5

Totals 14 221 214 26

Peptide L. 8 0 1 16 78 7 1 50:50 3 3 105 81 4 5 micro1 2 51 52 1 2 particles 3 5 151 107 2 5

Totals 25 423 318 27

Peptide L. 1 5 102 91 1 5

75:25 0 0 - 96 0 0 micro- 3 1 75 72 4 5 nαrrtr*l*»c *

Totals 10 177 259 15

one animal was killed after an injury

Table VI

Antigen Litter size¹ ±sem Reduction Litter size² ±sem Reduction

saline 6.0+1.0 6.0+1.0

Freund's 3.5±2.0 42% 7.0+0.0 0 adjuvant

50:50 6.3±1.0 0 6.3±1.0 0 m'particles

75:25 3.3±1.8 45% 5.0±0.6 17% m'particles

¹ in group as a whole

² in pregnant animals only

This shows that the highest number of fetuses (24 and 25) were obtained in animals immunised with peptide L-KLH in saline or 50:50 microparticles, the two weakest responding groups in Example 12. In contrast, rabbits immunised with L-KLH in Freund's adjuvant or 75:25 microparticles had mean litter sizes which represented reductions of 42% and 45% respectively. The litter sizes per pregnant doe were also reduced in the Group 4 animals.

These results suggest that in particular the 75:25 microparticle formulation for slow antigen release is an effective means of delivery leading to reduced fertility.

Claims

1. A method for controlling the fertility of a female mammal, said method comprising administering to said mammal an agent which stimulates an immune response, wherein the response includes the production of an element which interacts with uteroglobin of said mammal so as to reduce the fertility thereof.

2. A method according to claim 1 wherein said agent comprises a peptide or polypeptide which stimulates an immune response, said response including production of a binding element which specifically binds uteroglobin so as to reduce fertility of said female mammal

3. A method according to claim 1 wherein said agent comprises an expression vector which encodes a peptide or polypeptide which stimulates an immune response, said response including production of a binding element which specifically binds uteroglobin so as to reduce fertility of said female mammal, and expresses said peptide or polypeptide in vivo in said mammal.

4. A method acccording to claim 2 or claim 3 wherein the said binding elements comprise an immunoglobulin.

5. A method according to any one of claims 2 to 4 wherein the peptide or polypeptide comprises uteroglobin or a fragment thereof, or a variant or peptide mimetic of any of these, which is coupled to a carrier protein.

6. A method according to claim 5 wherein the polypeptide comprises full length uteroglobin of said mammal coupled to a carrier protein.

7. A method according to claim 5 wherein the peptide or polypeptide comprises one or more epitopic fragments of uteroglobin of said mammal coupled to a carrier protein.

8. A method according to claim 7 wherein the said fragment is from 4 to 25 amino acids in length.

9. A method according to claim 8 wherein the fragment comprises a species-specific motif.

10. A method according to any one of the preceding claims wherein the mammal is a rabbit.

11. A method according to claim 11 wherein the peptide or polypeptide comprises one or more of the following sequences:

MQMKKVLDS (SEQ ID NO 1)

LLGTPSSY (SEQ ID NO 3)

KEFEPDDT (SEQ ID NO 4)

LGTPSKEFEPDDTSLPQ (SEQ ID NO 2)

12. A method according to any one of the preceding claims wherein the carrier protein is selected from purified protein derivative (PPD), keyhole limpet haemocymin (KLH), bovine serum albumin (BSA) and ovalbumin (OVA).

13. A method according to claim 12 wherein the carrier protein is KLH.

14. A method according to any one of the preceding claims wherein the peptide or polypeptide is administered in the form of a pharmaceutically acceptable composition.

15. A method according to claim 14 wherein said composition comprises an adjuvant.

16. A method according to claim 15 wherein said composition is in the form of a microparticle formulation.

17. A method according to any one of claims 14 to 16 wherein the composition is adapted to allow slow release of said peptide or polypeptide over a period of time.

18. A method according to any one of claims 14 to 17 wherein the composition is adapted for oral administration.

19. A method according to any one of the preceding claims wherein said agent is administered in combination with other antigens or contraceptive reagents.

20. A peptide or polypeptide which is able to stimulate an immune response in a mammal, said response including production of elements which bind uteroglobin of said mammal so as to produce a reduction in the fertility of a female mammal.

21. A peptide or polypeptide according to claim 20 which comprises (a) uteroglobin or a fragment thereof, a peptide or polypeptide derived from uteroglobin, or a variant or peptide mimetic of any of these, and (b) a carrier protein.

22. A peptide or polypeptide according to claim 1 or claim 2 wherein the carrier protein is selected from purified protein derivative (PPD), keyhole limpet haemocymin (KLH), bovine serum albumin (BSA) and ovalbumin (OVA).

23. A peptide or polypeptide according to claim 21 or claim 22 which comprises rabbit uteroglobin as component (a).

24. A peptide or polypeptide according to claim 21 or claim 22 which comprises as component (a) a fragment of rabbit uteroglobin, a peptide or polypeptide derived from rabbit uteroglobin, or a variant or peptide mimetic of any of these.

25. A peptide or polypeptide according to claim 24 which comprises as component (a) a fragment of uteroglobin.

26. A peptide according to claim 25 wherein said fragment comprises one of the following amino acid sequences:

MQMKKVLDS (SEQ ID NO 1)

LLGTPSSY (SEQ ID NO 3)

KEFEPDDT (SEQ ID NO 4)

27. A peptide according to claim 26 which comprises

MQMKKVLDS (SEQ ID NO 1)

28. A peptide or polypeptide according to claim 25 which comprises two or more fragments of uteroglobin joined together.

29. A peptide according to claim 28 which comprises

LGTPSKEFEPDDTSLPQ (SEQ ID NO 2)

30. A nucleotide sequence which encodes a peptide or polypeptide according to any one of claims 20-29 or a part thereof, other than a sequence which encodes full length uteroglobin.

31. A nucleotide sequence according to claim 30 which encodes a part of the peptide or polypeptide including component (a).

32. An expression vector which comprises a nucleotide sequence according the claim 30 or claim 31.

33 A cell which has been transformed with a recombinant expression vector according to claim 32

34 A method for preparing a peptide or polypeptide according to any one of claims 20 to 29, comprising either (a) culturing a recombinant cell which has been transformed so as to express at least a part of said peptide or polypeptide, or

(b) synthesising a peptide or polypeptide using chemical synthesis, and thereafter if necessary, coupling the peptide or polypeptide obtained to a carrier protein

35 A pharmaceutical composition which comprises a peptide or polypeptide according to any one of claims 20 to 29 in combination with a pharmaceutically acceptable carrier

36 A composition according to claim 35 which further comprises an adjuvant

37 A composition according to claim 35 or claim 36 which is in the form of a slow release formulation

38 A composition according to any of claims 35 to 37 which is in the form of microparticles

39 A composition according to any one of claims 35 to 38 which is adapted for oral administration

40 An immunocontraceptive vaccine which comprises either a peptide or polypeptide which comprises (a) uteroglobin or a fragment thereof, a peptide or polypeptide derived from uteroglobin, or a variant or peptide mimetic of any of these, and (b) a carrier protein, or a recombinant virus vector which expresses said peptide or polypeptide

41 A method of producing an antibody which is specific for uteroglobin, which method comprises administering to a mammal, a peptide or polypeptide which comprises (a) uterglobin of said mammal or a fragment, derivative or variant thereof, and (b) a carrier protein and recovering antibodies produced.

42. An antibody obtainable by a method according to claim 41.