Note: Descriptions are shown in the official language in which they were submitted.
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WO 9X/00536 PCT!~BC/iOl-~o
Imrnunocontraceptive Methods and Peptide or Polypeptides for Use in these
~Iethods
The present invention relates to contraceptive me~hods erTective in m~nnm~l~ andparticular~y rabbits, to peptides and polypeptides u/hich are usefi~l in those
methods. tO processes for their production and to phar~naceutical compositions
cont~inin~ them.
The development of a contraceptive vaccine, particular~y for wild or feral species
0 of rn~m m~l~ such as wild rabbits would be desirable in order tO achieve a level of
control over the population levels of these m~m m~ls.
I,'teroglobin (UG) is the predorninant prote n in the uterine lumen of rabbits durinP the pre-
impiantation phase of pregnancy, accounting for up to 40-~0% of the protein content of the
uterine secretion in early prestnancy. It is a secreted prolein of ~ kD comprisinC a dimer
of two identical chains of 70 amino acids, linked by disulphide brid~es. The structure has
been fully charactensed (Morize I et al., J. Mol. Biol. (1987) 194: 725-7~9) and comprises
three helical regions interconnected by interhelical loops (Figure l).
~o Several prope~ des of UG have been identified, including binding of pro~esterone, inhibition of
infl~mm~tlon and immunosup~les~ e ef~ects by m~icin~ the aMi_enicity Ot sperrn and
embryos ~ithin the female tract.
The ~igh incidence of I~G in the pr~ L rabbit uterus impiies an important role for this
25 protein in pre~lancy. A~tisera to l;G have been raised in vaIious spe~ies such as sheep and
mice. It has be~n shown that anti- rabbit UG antibodies raised in chiç~Prt.~ and a~mini~red
passively to rabbits 2-~ days post-coi~n prevented impiantation (Krishnan R~. EYperientia
27: 95j-956, 1971).
30 rne applicants have r~ound that under certain circ-~m.~rlces. an iIr~nune response can be
srim~ ted in m~mm~l~ such as raboits, a ains~ utero~iobin or lo polypeptides or small
AMENDED SHEEt
. .
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peptides based upon uteroglobin. Furthermore, it has been found that the response can inhibit
fertility of the m~mm~l so giving rise to an immunncontraceptive
5 The present invention provides a method for controlling the fertility of a female m~mm~l j said
method Co~ g ~dmini~tçring to said m~mm~l an agent which stim--l~tes an immune
response, wherein the response includes the production of an element which interacts with
uteroglobin of said m~mm~l so as to reduce the fertility thereof
lo Suitable agents for use in the method comprises a peptide or polypeptide which stim~ tes an
immlme response, said response inçlutling production of a binding element which specifically
binds uteroglobin so as to reduce fertility of said female m~mm~l or an t;~,ression vector
which encodes a such a peptide or polypeptide and eA,ulesses said peptide or polypeptide in
vivo in said m~mm~l
As used herein the term 'polypeptide' is intçnded to enco~ ,ass proteins.
Binding çlçm~nt~ will generally comprise immllnoglobulins and in particular antibodies. ln
order to achieve a contraceptive effect, it may be necessa- y to deliver the peptide or
2n polypeptide using an applu~liale delivery strategy as is conventional in the art
Preferably, the method will collll,.ise ~tlmini~tration of a peptide or polypeptide which
stim--l~tes an immune response, said response including production of a binding element
which bind uteroglobin and reduce fertility of said female I~ A
Novel peptides or polypeptides for use in this method, which are able to stim~ te an imm--ne
response in a m~mm~l, said response having an effect on uteroglobin so as to reduce the
fertility of a female m~mm~l, form a further aspect of the invention
30 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
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m~mm~l Thus for ~A~"~plc, where the target m~mm~l is a rabbit, the polypeptide used is
preferably rabbit uteroglobin or a fragment thereof, which has been rendered immllnngenic for
example by coupling to a carrier.
5 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 immllnic~tion methods.
Techniques for using such antibodies in detection and diagnosis are well known. They may
o include ELISA techniques, or the use of labelled antibodies or anti-antibodies, for example
gold labelled antibodies. Both col"pelilive and direct assays may be formulated in a
conventional manner.
Thus the invention also provides a peptide or polypeptide which comprises uteroglobin
15 fr~gment, 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 l.l~ l 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 fr~m~ntc for example of from 4 to 25, suitably from 8 to 17 amino acids in length.
Indeed, small fr~gmPnt.c are plerelled as they may be easier to synthPcice using chemical
means and thel eru, ~ they may be supplied in greater quantities at reduced cost. In addition,
2~ 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
fragmentc are selected so that they are largely species specific and in particular do not cross-
react with other species found in similar env"unl~lenls such as the hare.
Several fr~gm~nts 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 tAa"lple of such
-
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a peptide, derived from the three interhelical regions is given hereinafter and de~ign~ted
peptide "L".
As used herein, the term "variant" means that the peptide or polypeptide has a sequence which
s is similar to that of uteroglobin or fragments thereof, but wherein one or more amino acid
residues are di~~ l. The changes do not alter function of the peptide or polypeptide in
terms of its ability to produce an immune response in a female m~mm~l which affects the
fertility of that m~mm~l although the extent of that response and the resultant affect may be
at a di~elenl level. For ~Aal.lple, peptides or polypeptides which are 60% homologous to the
o native sequence, suitably more than 80% homologous and pl~lably more than 90%
homologous to the native sequence and which have similar gross biological pl opel lies would
constitute "variants" .
The expression "peptide mimPtic" used herein refers to peptides or polypeptides which are
15 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. Th~l ~rOI ~, 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
20 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 m~mmal. A native protein such as uteroglobin or fr~gm~nt.~ would
not be seen as foreign by its natural host. In order to induce, an immune response, it is
25 necess~ry 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 hllpol 1~lll factor in detel ll.millg the level and
30 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 pl ~r~ d carrier
as col~.paled to OVA.
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In a plc~ed embodiment, the uteroglobin is rabbit uteroglobin, which has an effect of
reducing rabbit fertility.
5 Thus a particularly p~ led embodiment ofthe 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 fr~gm~nt.c of rabbit uteroglobin are employed. The
10 skilled person would be able to test using routine methods, for example as illustrated
hereinafter, whether any particular fragment (or combination of fragm~nts), 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
15 with uteroglobin. Selection of suitable delivery techniques and formulations to ensure
optimum contraceptive effect can also be determined using conventional techniques for
fertility ~sess,-.enl, for example as illustrated here,n&ner.
It has been found that in particular, peptides derived from the third helix of rabbit uteroglobin
20 have the desired biological activity. One such peptide comprises a nonapeptide of sequence:
MQMKKVLDS (SEQIDNO 1)
which has been de.cign~ted 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:
LGTPSKEFEPDDTSLPQ (SEQIDNo2)
which has been decign~ted for the purposes of this application as peptide "L".
-
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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
s 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 t;rre~;led
chemically in order to obtain shorter or modified fragments.
Yet a further alternative is production using I ~combinalll 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 synthesi~ing techniques such as automated nucleic acid
1 5 synthesisers.
The nucleic acid is then introduced into an al)p-op-iate 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
20 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.
25 Novel nucleic acid sequences, replication vectors or plasmids, transformed cells and processes
for ~ epa ing the peptides or polypeptides form further aspects of the invention.
Using reco-l~inatll 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
30 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
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WO 98/00536 PCT/GB97/01740
linkers such as sulphosucrinimidyl 4-(N-maleidimethyl)cyclohexane-l-carboxylate or bis-
diazotised-o-toluidine.
When used as an immllnocontraceptive~ the peptides or polypeptides are suitably a-lmini~tered
in the form of a pharmaceutical composition which further comprises a pharm~ceutically
accel)lablc 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 potenti~tes the
0 imrnune response. Such adjuvants include Freund's complete and incomplete adjuvant,
minium compounds such as phosphate and hydroxide, mineral oils such as squalene or
blodegradable peanut oil, or muramyl dipeptide which may be incorporated into the mineral
oll.
Various methods of a(1mini~tration 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 100,ug 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
200~1g/animal for the initial dose, with subsequent doses of 100~g/animal. However, when
using small peptides of the invention, dosages of the complex with PPD were suitably
500,ug/animal for the initial dose, with subsequent doses of 250~g/animal.
Instead of a(lmini~tering the peptides or polypeptides directly, they could be produced by a
live vaccine. In this case, a live vector, for example an ~ttenll~ted virus, such as an attenll~ted
vaccina virus, is tran~rol med such that it expresses the antigenic peptide or polypeptide-carrier
30 conjugate.
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However, preferably, imnlllnocontraceptive 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
5 immunostim~ tory complexes or "ISCOMS" (see for example Morein B and Akerblom L
(1992) in Reconlbina,,l 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.
o The peptides or polypeptides of the invention may be ~mini~tered 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 applop,iate target for
5 immunocontraception and that fertility is significantly reduced as a results of UG
immllni~tion. 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
20 acco~panying drawings in which:
Figure 1 is a computer derived image of rabbit UG, showing the so-called "~ntifl~mmin"
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;
30 Figure 3 is a graph showing the results of an ELISA assay of antiserum to peptide F, the
~ntifl~mmin peptide; the upper graph shows antiserum tested against the peptide, and the
lower graph shows antiserum tested against native uteroglobin;
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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 immuni~ed with a variety of
antigens and an irrelevant control peptide and Figure 5b shows the incidence of resorptions
recorded in the experimental groups;
lO Figure 6 shows the time course of primary response to a peptide of the invention incorporated
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 ofthe
invention in various formulations.
Example 1
Preparation of Rabbit UG
20 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 (0.1 5M), the washings centrifuged and the SUp~lllalalll~
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
Al.56m column (Bio-Gel Laboratories Inc), eluting with PBS, pH 7.2 at 12 mls/hour,
collecting 3 ml fractions (Figure 3a). Fractions cont~ining UG were identified by SDS-PAGE,
30 pooled, freeze dried, redissolved and dialyses against PBS in CelluSep T2 dialysis membrane,
MW cut off 8-1 OkD.
...... , ~
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In
The UG was further purified by FPLC gel filtration on Superdex 75 HR10/30 column. The
resultant UG prepal~lion did not contain other proteins detectable by gel electrophoresis.
Q~l~ntit~tion of UG was by measurement of absorbance at 205nm.
5 Example 2
Plt;~a~lion of Recol~bil~anl UG7
Isolation techniques such as that described in Example 1 produced relatively small yields of
protein. The vector pDS-UG7 which induces high level ~pl e~ion of recolllbinanl rabbit
uteroglobin in bacteria (W. Peter et al., Protein Engineering (1989) 3: 61-66) was obtained.
o The expressed recc,lllbin6nl UG (rUG&) forms stable dimers and binds progesterone
inrli.~tine~ h~hly from native UG. E. coli strain W3 110 was ll~llsrol'''ed with pDS-UG7 and
protein ~I,ression induced with isopropylthiogalactoside (IPTG). Bacteria were centrifuged,
suspended in water and extracted by ultrasonic disruption. After centrifugation at I O,OOOx~
for 15 minllte.c, the soluble fraction was made to IOOmM Tris-HCl (pH 7.5), 150mM NaCI
15 and IOmM 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, Smg/ml; Aprotinin 1%v/v).
The protein extract was gel filtered on a Bio-Gel Al.5m column in PBS co.~ ;ng 0.1%
20 sodium a~ide and I OrnM DTE, and fractions cont~ining rUG7 identified by gel analysis.
These were pooled, concentrated by freeze drying and further purified by FPLC on a
Superdex 75 column (Pharrnacia). The identity of rUG7 was determined by COnlj~&l ison with
molecular weight standards and highly purified UG and confirmed by amino acid sequence
analysis of the purified protein.
2s
Example 3
Plel,al~lion of Peptide Antig~n.c
The following peptides were prepared using conventional microchemical techniques:
30 1. Peptide L, a peptide based on the three inter-helical loops of UG, Leu-l 5 to Ser-l9,
Lys-26 to Thr-33, and Ser-47 to Gln-50, synthesises as a single 17-mer peptide of
sequence:
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LGTP SKEFEPDDT SLPQ (SEQIDNO2)
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 (SEQIDNO3)
lo Loop2- KEFEPDDT (SEQIDNO4)
Loop3- LDSLPQTT (SEQIDNO5)
3 . Peptide F, a nonapeptide Met-39 to Ser-47 which forms the major part of the third
ls helix of UG and has sequence similarity to the ~ntifl~mmin.~ (L. Miele et al., Nature
(1988) 335: 726-729). The sequence ofthis peptide is:
MQMKKVLDS (SEQIDNO l)
Example 4
Conjugation of Protein or Peptides to Carriers
Peptides were conjugPted 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 minlltes, the pH adjusted to pH 6.0 and activated
PPD separated by gel filtration from uncoupled linker. Peptides of Examples I, 2 and 3 were
coupled overnight to activated PPD under nitrogen at pH 7Ø 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.
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12
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
5 Tmmllni~tion Studies
Tmm~lni~tion of BCG-primed rabbits using the peptide or protein conjugates from Example 4
was carried out with a minim~lm of two injections. Primary injections of antigen peptide-PPD
were given in incomplete Freund's adjuvant both intr~ml~sc~ rly and subcutaneously and
subsequent booster injections were given subcutaneously with a 3 week interval between
0 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). Ofthe three peptides based
on loop sequences? Loop 2 antisera were strongly cross-reactive against UG, Loop 1 wealcly
20 cross reactive and Loop 3 antisera non-cross reactive.
Example 6
Effect of lmmuni~ation on Fertility
In a fertility trial, groups of 5 female rabbits were imml~ni.~ed prior to mating and their
25 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 imm~lni.~ed with the UG-PPD conjugate or peptide F-PPD conjugateshowed 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, co,llpaled with 6.4 for those immlmi~ed with the
30 control peptide. It was significant that there were a high number of fetal resorptions in the
animals immlmi.~ed with peptide F (Figure 5b). This suggests a late effect on fetal viability
resulting from immlmi.~tion
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W098/00536 13
Example 7
Fertility Trials
Groups of 10 female dutch rabbits were immuni~ed with the following llea~lllellts:
s
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).
lo Primary imml~ni.c~tions 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 immllni.c~tion with
peptide F.
Tmmuni.ced female rabbits were introduced to a buck rabbit on two consecutive days for
mating. Fertility was ~ses.sed 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 1 and II.
..
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14
Table I
Antigen No offetuses Weight of fetuses Weightofuterus Corporalutea
L R L R
Control 0 o - 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
I+IR 4+1R 84 137 2 6
4 3 111 128 4 3
2+1M 59 71 5 5
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
O O - - O O
O O - - O
0 0 - 5 2 2
0 0 - 7 3 6
4* 222 nd
7* nd nd
2 142 79 5 2
Totals 35 768 313 43
UG 0 2 71 38 3
2 2 121 83 5 4
2 0 58 47 4 2
O O - - O O
l+lR 0 27 29 1 4
3 4 79 99 3 4
0 0 - 7 2 2
0 0 - 9 4 4
O O - 10 1 0
0 2 41 49 2 2
Totals 19 397 371 48
* live births
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Table II
Antigen Litter size' +sem Reduction Litter size2 +sem Reduction
Control 4 4+0.96 5 5+0 78
PeptideF 3.5+1 1 21% 5 8+095 o
UG 1.9+0.83 57% 3 2+0.83 42%
lo l in group as a whole
2 in pregnant animals only
The results demonstrate a marked reduction in fertility of rabbits immlmi~ed with UG: in the
control group, 8 ofthe 10 rabbits became pl~snalll and produced 44 fetuses (mean litter size
of 4.4 + 0.96), whereas in does imm~.ni~ed 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-imm-lni~ed rabbits Among the plegnal1l
does, litter size was reduced from 5 5 + 0 78 in the control group to 3 2 + 0 83 in the UG
immllni~ed group, a reduction of 42% The fact that only 19 out of 48 corpora lutea (39.6%)
gave rise to implantations in the UG-imm--ni~ed rabbits COn~l)a~ ~d 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 immllni.~ed against peptide F, the number plegnanl was again reduced to 6, and the
2s total number of fetuses was 35 (mean litter size 3.5 + I .1), a reduction of 21%.
Statistical analysis of the results in Table II showed that the overall litter size of the UG-
immllniced does (1 9 + 0 71) was significantly smaller than that ofthe 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
30 program (p=0.052) Coll.palhlg only the pregnant does of these two groups, the litter size of
the UG-imm--ni~ed animals (3.2 + 0 83) was also significantly reduced compaled with the
controls (5.5 + 0 78) (p=0 66) In contrast, the litter size ofthe peptide F imml-niced group
CA 022~9237 1998-12-24
W 098/00536 PCT/GB97/01740 16
(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 dirrele.ll.
This trial clearly demonstrated that imm~lni~tion against UG in particular is effective as an
5 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 ~nim~l~; moreover, it was statistically significant at the
95% level.
o Example 8
Passive immlmi~tion with sheep anti-UG antibodies.
A serum was raised by immllni.~tion 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
15 from a nonimm-lni~ed sheep was pl~pared 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, ~ days before and at 4, 11, and 18
days after mating; the control group received 4 injections of normal sheep IgG to the same
20 amount. Animals were autopsied on day 25 of p,~gl-ancy.
The results of this trial are given in Tables III and IV.
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Table III
Injected No offetuses Weightof fetuses Weight of uterus Corporalutea
L R L R
Normal 5 S 145 97 5 5
sheep IgG 0 0 - 9 1 2
7 2 1 16 143 7
2+1R 2 43 87 3 2
0 0 0 14 7
Totals 24 304 350 34
Anti-UG 2 3 96 101 2 S
sheepIgG 0 0 - 13 0 0
0 0 - 9 0
3 4 129 123 3 4
0 0 - 7 0 0
Totals 12 225 253 5
Table IV
o Antigen Litter size'_sem Reduction Litter size2_sem Reduction
Normal 4.8+21 8.0_1.2
sheep IgG
Anti-UG
sheep IgG 2.4_1.5 50% 6.0+0.6 25%
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 ofthe 5 rabbits were ,Ole~n&
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and produced 12 fetuses (mean litter size 2.4 + 1.5), whereas in the control group, 3 ofthe 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 red~lcin~ fertility.
Example 9
o 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
15 Antigen Litter size' +sem Reduction Litter size2 +sem Reduction
Controls 4.53+0.91 6.18+0.75
UG immlmi~ed 2.07+0.66 54% 3.87+0.79 37%
l in group as a whole
2 in plegnalll animals only
This shows that the effect of UG immnnic~tion on litter size is st~ti!~tic~lly highly significant
(Table V). Including both ~legnanl and nonpregnant animals in each group, the litter size in
the UG immllni~ed group was 2 07 + 0.66 compaled 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 immnnised 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 di~elenl between control and imm~mi~ed groups, the
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19
results demonstrate that the effect of UG imm~lnic~tion is to reduce mean litter size
significantly.
Example 10
5 Preparation of Biode~radable Microparticles
Conjugates as prepared in Example 4 above were incorporated into microparticles by mixing a
1 5mg/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
lo 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. lM sodium
5 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 1 1
Antibody responses to peptide L after imm-mi.cfltion in biodegradable microparticles
20 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
immlmi~tion 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
25 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 ind~lced
quickly, peaking at 20 days, and then declining; the response to L-KLH was stronger than that
30 to L-KLH.
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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 microparticle-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 11, four groups of 16 rabbits each received L-KL H in a
di~~ 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 di~l ~..l 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 lesponse with a sust~ined peak between days 20 and 60 and deçlining thereafter.
Example 13
Fertility Trials with Animals followin~ immllni~tion 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.
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Table VI
Antigen No offetuses Weight of fetuses Weight of uterus Corporalutea
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
3 69 55 3 3
Totals 24 340 315 34
Peptide L, 0 0 - 11 1 7
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 0 116 78 7
50:so 3 3 105 81 4 5
micro- 1 2 S l 52 1 2
particles 3 5 I S l 107 2 S
Totals 25 423 318 27
Peptide L. 1 5 102 91 1 5
75 :25 0 0 - 96 o 0
rnicro- 3 1 75 72 4 5
particles *
Totals 10 177 259 15
* one animal was killed after an injury
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Table VI
Antigen Litter size' +sem l~eduction Litter size2 +sem Reduction
s
saline 6.0+1.0 6 0+1.0
Freund's 3 5+2.0 42% 7.0+0.0 o
adjuvant
50:50 6.3+1.0 0 6 3+1.0 o
m'particles
75:25 3.3+1.8 45% 5.0+0.6 17%
m'particles
' 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
20 immllnised with peptide L-KLH in saline or 50:50 microparticles, the two weakést responding
groups in Example 12. In contrast, rabbits imm~lni~ed with L-KLH in Freund's adjuvant or
75:25 microparticles had mean litter sizes which leplescnled reductions of 42% and 45%
lespe~ ely. The litter sizes per pley,na~l doe were also reduced in the Group 4 animals.
25 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.
