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  • lorsque la demande peut être examinée par le public;
  • lorsque le brevet est émis (délivrance).
(12) Brevet: (11) CA 2137363
(54) Titre français: UTILISATION DE GLYCOPROTEINES DE LA MEMBRANE PELLUCIDE A DES FINS D'IMMUNOCONTRACEPTION
(54) Titre anglais: USE OF ZONA PELLUCIDA GLYCOPROTEINS FOR IMMUNOCONTRACEPTION
Statut: Réputé périmé
Données bibliographiques
(51) Classification internationale des brevets (CIB):
  • A61K 39/00 (2006.01)
  • A61K 9/127 (2006.01)
(72) Inventeurs :
  • BROWN, ROBERT (Canada)
  • KIMMINS, WARWICK (Canada)
  • MEZEI, MICHAEL (Canada)
  • POHAJDAK, BILL (Canada)
(73) Titulaires :
  • IMMUNOVACCINE TECHNOLOGIES INC. (Canada)
(71) Demandeurs :
  • DALHOUSIE UNIVERSITY (Canada)
(74) Agent: SMART & BIGGAR
(74) Co-agent:
(45) Délivré: 1999-06-15
(86) Date de dépôt PCT: 1993-06-07
(87) Mise à la disponibilité du public: 1993-12-23
Requête d'examen: 1997-11-03
Licence disponible: S.O.
(25) Langue des documents déposés: Anglais

Traité de coopération en matière de brevets (PCT): Oui
(86) Numéro de la demande PCT: PCT/CA1993/000239
(87) Numéro de publication internationale PCT: WO1993/025231
(85) Entrée nationale: 1994-12-05

(30) Données de priorité de la demande:
Numéro de la demande Pays / territoire Date
892,807 Etats-Unis d'Amérique 1992-06-05

Abrégés

Abrégé anglais



A vaccine for the immunocontraception of mammals is described. The vaccine consists of zona pellucida antigens and an
adjuvant encapsulated in a liposome delivery system. The liposome delivery system allows for the slow release of antigen resulting
in a prolonged immune response. In particular, after a single injection of the vaccine, levels of anti-zona pellucida antibodies
were detected for up to 22 months. Thus, the vaccine according to the present invention is effective after a single dose and is
therefore very useful in immunocontraceptive protocols.

Revendications

Note : Les revendications sont présentées dans la langue officielle dans laquelle elles ont été soumises.



-19-
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:


1. A parenteral vaccine composition for the
immunocontraception of a mammal which comprises a zona
pellucida derived antigen and a suitable adjuvant in a
liposome formulation.



2. A vaccine composition according to claim 1 wherein
said zona pellucida antigen is ZP3.



3. A vaccine composition according to claim 1 wherein
aid zone pellucida antigen is solubilized intact zona
pellucida.



4. A vaccine composition according to claim 1, 2 or 3
wherein said zona pellucida antigen is porcine derived.

5. A vaccine composition according to any one of
claims 1 to 4 wherein said adjuvant is Freund's adjuvant.



6. A vaccine composition according to any one of
claims 1 to 5 wherein said liposome system is freeze-dried.


7. A method of preventing fertilization in a mammal
which comprises administering an effective amount of a
parenteral vaccine composition according to any one of claims
1 to 6.


-20-
8. A method according to claim 7 wherein said
composition is administered once as a single dose.


9. A method according to claim 7 or 8 wherein said
mammal is selected from seals and rabbits.


Description

Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.


WO 93/25231 213 7 3 6 3 PCI'/CA93/00239




IJSE OF ZO~A PEI.LtJCIDA GLYCOPR~ NS FOR I~IJNOCON~RP,~PTION
FIELD OF THE INVENTION
The present invention relates to a vaccine com-
position for the immunocontraception of mammals.
5 BACKGROUND OF THE INVENT I ON
There is a real need for population control in
several species of domestic and wild Anl~ls~ Methods such
as surgical sterilization, or more drastically, culling are
generally not acceptable or even allowable in most
countries. For example, in Canada the culling or harvest-
ing of seals was prohibited in the early 1980's, resulting
in an increased seal population from 10,000 in 1978 to
approximately 45,000 at present. Increases in harp seal
populations have been much greater. Unfortunately, the
15 increasing population of seals is eating away at the dimin-
ishing fish stocks which poses serious problems for the
fishing industry. The seals also contain parasites such as
seal worms that they pass on to the fish. In 1986 it was
estimated that the cost to the fishing industry of removing
seal worms from fish by hand was upwards of S30 million a
year. Therefore, it is highly desirable to develop an
effective form of contraception in ~mm~ls, such as seals,
in order to effectively control the population growth of
certain m~mm~ 1 S .
One form of contraception for m~m~l S has in-
volved immunocontraception using glycoproteins isolated
from the zona pellucida, a covering which surrounds
oocytes. The zona pellucida glycoproteins (hereafter
referred to as ZP) provide an attachment site for sperm.
Immunocontraception with ZP results in the production of
antibodies to ZP which cause (a) an alteration of the
nature of the ZP membrane of ova, thereby inhibiting sperm
entry, (b) an inhibition of implantation of fertilized ova
into the uterus and (c) decreased ovarian follicular
differentiation (Henderson, C.J., M.J. Hulme and R.J. Ait-
ken. 1988. Contraceptive potential of antibodies to the
zona pellucida. J. Reprod. Fert. 83: 325-343).
SUt~a 1 l l IJTE SHEEl-

WO93/2523l ~13 7 3 6 ~ PCT/CA93/00239


Immunocontraception has been induced with both zona
pellucida glycoproteins and epitopes of these glycoproteins
which have been sequenced, synthesized and coupled to
carrier proteins (Millar, S.E., S.M. Chamow, A.W. Baur,
C. Oliver, F. Robey and J. Dean. 1989. Vaccination with a
synthetic zona pellucida peptide produces long-term
contraception in female mice. Science 246: 935-938). The
use of ZP glycoproteins for immunocontraception has several
advantages over other contraception methods. Firstly, ZP
glycoproteins are unique to the female reproductive system
and therefore, anti-ZP antibodies likely have little or no
effect on other tissues. Secondly, the infertility caused
by anti-ZP antibodies is reversible, although, hyper-
immunization may cause permanent sterility.
Previous studies have shown that in order to
effect immunocontraception, multiple injections of ZP were
necessary (Kirkpatrick, J.F., I.K.M. Liu and J.W. Turner.
1990. Remotely-delivered immunocontraception in feral
horses. Wildl. Soc. Bull. 18: 326-330). Multiple injec-
tions are clearly not practical for wild populations as it
entails recapturing the same wild animal each time an in-
jection is necessary. Multiple injections are also cumber-
some in any situation.
Therefore, it is desirable to develop an immuno-
contraceptive vaccine which would be effective for longperiods following a single injection in an efficient
delivery system.
Liposomes have been used to carry drugs to sites
of inflammation and infection or in some cases tumours.
Liposomes are microscopic spheres composed of either a
single or multiple concentric bilayer sheets, and range in
size from a nanometer to several micrometers in diameter.
These bilayer sheets can be formed from a wide variety of
phospholipids in varied formulations. Cholesterol can be
included in the bilayer in order to increase the bilayer
strength and reduce the leakage of materials encapsulated
within the entrapped aqueous interior. A vast array of
SUBS 111 IJTE SHEE~

-

- 3 -
compounds can be associated with liposomes, including small
molecules, drugs, proteins, and nucleic acids. Liposome-
associated compounds can be encapsulated within the aqueous
interior of the liposome (i.e. between the bilayer sheets),
integrated into the bilayer, or adsorbed or attached to the
bilayer surface. The location depends upon the properties of
the associating compounds as well as the procedures used for
the formation of the liposome.
A liposome based vaccine system has been described
for immunization against human malaria (Fries et al, 1992.
Liposomal Malaria Vaccine in humans: A safe and potent
adjuvant strategy. Proc. Natl. Acad. Sci. USA. 89: 358-362).
In this system, a recombinant malaria protein derived from
Plasmodium falciparum was encapsulated into liposomes and
injected into male volunteers. However the results indicate
that at least three injections of the vaccine were required
in order to produce an elevated antibody response.
SUMMARY OF THE lN V ~N'L ION
The present invention relates to an
immunocontraceptive vaccine preparation which comprises zona
pellucida antigens incorporated into a liposome delivery
system. The liposome system effects the slow release of
antigen resulting in an extended period of antibody
production and thereby an extended period of contraception.
Therefore, the present invention provides a method to achieve
immunocontraception of mammals using a single injection of
zona pellucida glycoproteins.
Accordingly, the invention provides a parenteral



74268-12(S)

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vaccine composition for the immunocontraception of a mammal
which comprises a zona pellucida derived antigen and a
suitable adjuvant in a liposome formulation.
The invention further provides a vaccine
composition capable of inducing the production of antibodies
to a zona pellucida antigen, said composition comprising a
zona pellucida derived antigen incorporated into a liposome
system.
In one embodiment of the present invention, the
liposome vaccine composition is freeze-dried and incorporated
into a BallistiVet biobullet. Such an embodiment makes the
vaccine easier to deliver to the animal.
In another aspect, the invention provides a method
of preventing fertilization in a mammal which comprises
administering an effective amount of the above-described
vaccine.
DETAILED DESCRIPTION OF THE lNv~NLlON
Isolation and purification of zona Pellucida qlycoproteins
Zona pellucida glycoproteins were isolated and
purified as described by Yurewicz (in Yurewicz, E.C., A.G.
Sacco, and M.G. Subramanian, Structural Characterization of
the Mr = 55,000 Antigen (ZP3) of Porcine Oocyte Zona
Pellucida, The Journal of Biological Chemistry 262:564-571
(1987)). Porcine ovaries were homogenized and the oocytes
recovered from the homogenate by sieving through nylon
screens of decreasing pore size (500, 350, 200, 175, 100 and
40 ~m). The oocytes were homogenized with a glass-teflon*
*Trade-mark



~ ~ 74268-12(S)


- 4a -
apparatus and the homogenate was passed through a 40 ~m
screen to collect the fractured zonae. The fractured zonae
were washed with buffer and recollected on the 40 ~m screen.
Zona pellucida glycoproteins were solubilized by incubating
the zonae in a water bath (73~C for 20 minutes). The
fraction obtained (solubilized intact zona pellucida
glycoproteins, SIZP) was shown to be at least 95% pure by
comparison to a reference standard of ZP using an ELISA
assay.
ZP3 was purified from SIZP as described by Yurewicz
et al (1987). ZP3 is one of three glycoproteins that make up
the mammalian zona pellucida. ZP3 is the major
macromelecular component of the oocyte zona pellucida and has
been shown to be the receptor for sperm.
Porcine ZP has been used to effect
immunocontraception in a variety of mammals. Porcine ZP
pellucida was used in the present studies for several
reasons. Firstly, a comparison of the reactivity of ZP from
five mammalian




~'
74268-12(S)

WO93/25231 213 7 3 6 3 PCT/CA93/00239

" ,_




species to rabbit antiserum against porcine ZP indicated
that pig ZP was recognized best followed by dog ZP while
rat and cat ZP reacted poorly (Maresh, G.A. and B.S. Dun-
bar. 1987. Antigenic comparison of five species of
mammalian zonae pellucida. J. Experimental Zoology 244:
299-307). Since seal and dog are closely related, (Berta,
A., C.E. Rae, A.R. Wyss, 1989, "Skeleton of the oldest
known pinniped Enaliarctos mealsi", Science .244: 60-62),
anti-porcine ZP antibodies will bind to seal zonae.
Secondly porcine ZP can also be easily obtained from
slaughterhouse pigs. Thirdly, pigs ovulate multiple
oocytes and thus provide a rich source of zona pellucida.
EXPERIMENT 1
Production of Anti-ZP Antibodies in Rabbits
Protocol.
Rabbits were injected i.m. (two rabbits for each
treatment, details of each treatment are given in Table 1)
as follows (a) ZP in three injections at monthly intervals,
(b) ZP with Freund's complete adjuvant plus two boosters
with ZP in Freund's incomplete adjuvant at 4 and 8 weeks
and (c) ZP with muramyl dipeptide adjuvant (Sigma chemi-
cals). In a second series, rabbits were immunized as above
but ZP, plus adjuvant where applicable, were encapsulated
in liposomes and in all cases in the second series only a
single injection was administered. Preimmunization serum
samples were taken from all rabbits. Titers of antibodies
were measured by Elisa assay using both SIZP and ZP3 as
antigen.
Results.
The results of this study are illustrated in
Table 1. Immunization of rabbits with ZP (no adjuvant)
raised a low antibody titer after the third injection. A
single injection of ZP in liposomes with no adjuvant raised
no detectable antibodies. Use of muramyl dipeptide ad-
juvant did not stimulate antibody production regardless of
being encapsulated in liposomes. Use of Freund's adjuvant
stimulated antibody production to high levels after 69 days
SU~ ITE SHEET

WO93/25231 ~ 13 7 3 6 3 PCT/CA93/00239


when the antigen was administered by 3 injections 4 weeks
apart or as a single injection in liposomes. However,
titers were higher using the former procedure. Measurement
of antibodies directed specifically against ZP 3 indicated
that antibody titers were similar after three injections
without the use of liposomes as they were with a single
injection with liposomes.
Therefore, these results indicate that a vaccine
preparation containing ZP in combination with Freund's
adjuvant and encapsulated into a liposome system can induce
significant anti-ZP antibody levels for extended periods of
time after a single injection.




SUB~ 111 LITE SHEET

Table 1 ~ I 3 ~ 3 ~ 3 ~
~_,ffect of Encapsulating ZP in Liposomes
on Anti-ZP Antibody Production in Rabbits
~ntl-ZP tlter~
Time Ag a b c d e f
~day~)
PRE SIZP 0 ~ 0 0 0 0 0 0 0 0 0 0
2~ SIZP 2 0 12 2 0 0 0 0.5 22 36 0 0.7
SIZP 12 3 96 6~ 2 1 0 0 50 S0 0.3 0.6
69 SIZP 24 24 1~4 130 - - - - ~4 ~5
69 ZP316 15 97 46 - - - - 74 64 - -
A value of 100~ indicates the ~erum being tested has the ~ame
tlter a~ a pooled ~ta~dard serum from rabbit3 rendered i~fertile
by immunizat~on wlth SIZP.
a - each rabbit wa~ lnjected w~th SIZP ~20 ~g) in 0.5 ml ~aline.
b - each rabbit wa~ inje~ted f~rqt with SIZP ~20 ~g in a m~xt~re of
~aline (0~25 ml) and Freund'~ complete adjuvant (0.25 ml)
~ollow~d by two booster~ o~ SIZP (20 llg~ a mixtur~ o~ e
~0.25 ml) and Freund' 9 incomplete adjuvant ~0.25 ml).
c - each rabbit waq injected with S~ZP ~20 ~g) with muramyl dipeptide
(5 ~g) in saline (0.5 ml).
d - eac~ rabbit wa~ injected with SIZP ~20 ~g) encap~ulated in
lipoaome~ coneaining phospholipon 90G ~Nattermann Phospholipid
Co., Cologne, Genmany, 0.1 g), choleaterol ~0.01 g) and ~aline
(0.5 ml). Lipoaomes were prepared ac~ordinq to U.S. patent
4,4~5,054. Liposomal
product~ containing ZP and adj~vant~ however can be prepared by
many other methods presently known and used for manufacturing
liposomea.
e - each rabblt was injected w~th SIZP ~20 ~g) encap~ulated in
llpo~ome~ a~ for d ~0.25 ml) in ~eund'~ complete ad~uvant
~~ 25 ml).
f - each rabb~t wa~ ~njected wlth SIZP ~20 ~g) encapsulated in
l~poaomet a~ for d ~0.5 ml) conta~n~ng m~ramyl d~peptide ~5 ~g).
PRE- pre~mmunization.

EXP~RIM~NT 2
Immunization of CaPtive Seals
Protocol.
Captive female grey seals (13) were divided into
six groups of two animals each, except for group d which
comprised three animals. The groups were immunized with a
single injection i.m. as follows:
! (a) zP (15 ~g) in liposomes with ~reund's complete
ad~uvant (FCA~


74268-12(S)




... - .. . ..

8 ' A~137363

(b) ZP (90,ug) in liposomes with FCA
(c) ZP (90~g) in FCA
(d) ZP (9~1~9) in liposomes with TiterMax
(e) ZP (90 /lg) in TiterMax
(f) ZP (90 llg) in liposomes with BCG adjuvant in water
All animals received an injection having a volume of 1.0 ml.
Bacillus Calmette-Guerin or BCG is one component of FCA. Liposomes were
formulated as previously described. TiterMax (0.5 ml) was mixed with
saline (0.5 ml) containing SIZP or liposomes (0.5 ml) containing SIZP as
described by the manufacturer. TiterMax (Cytrx Corporation, Norcross,
Georgia) is a new adjuvant system which claims to induce higher titers of
antibodies than FCA with a single injection while being less toxic to animals.

WO 93/25231 ~13 7 3 6 3 PCT/CA93/00239

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SUBS 111 ~JTE SHEET

WO93/25231 21 3 7 3 6 3 PCT/CA93/00239


Results
The results of the immunization of the seals are
shown in Table 2. The 1st and 2nd line of each group
refers to results for two seals receiving the same treat-
ment, that is, each formulation was tested in two seals,except for the ZP/TiterMax/liposomes formulation which was
tested in three seals. As can be seen from this table, the
anti-ZP titer was greatest for groups (a) and (b) wherein
zona pellucida was incorporated into a liposome system.
Antibody titers were detected up until 22 months from
immunization in the three seals from groups (a) and (b).
(The fourth seal was returned to the wild at 11 months.)
Without liposomes, group (c), antibody titers dropped off
after 5 months. Substituting BCG for Freund's complete
adjuvant was not effective in eliciting high titers
(group f). The new adjuvant system, TiterMax, was also not
effective (groups (d) and (e)).
Therefore, the results of this study demonstrate
that incorporation of the ZP and adjuvant into a liposome
system results in a prolonged anti-ZP response after a
single injection.
EXPERIMENT 3
Immunization of Harbour Seals
Twelve harbour seals, divided into three groups
of four, were immunized with ZP in a single injection
(Table 3). The seals were immunized immediately after
giving birth. Samples were taken in the two or three week
period until the seals left the pupping area. The results
are shown in Table 3. Immunization (with 30 and 90 ~g ZP)
of harbour seals that had just given birth resulted in
anti-ZP titers of only 9% (range 6 to 12%) of immunocontra-
ceptive levels after 3 weeks. As discussed previously,
anti-ZP antibodies can act as an immunocontraceptive by
inhibiting sperm penetration of ova or by inhibiting
implantation of fertilized ova into the uterus. Since
seals breed about 3 weeks after giving birth, it is
possible that the above levels of anti-ZP antibodies would

SUB~ 111 IJTE SHEET

W O 93/25231 ~13 7 ~ 6 3 PC~r/CA93/00239
-


11
not affect sperm penetration of the oocytes. Immunization
directly after giving birth may, however, have some effect
on the implantation of the embryo sinc~ seals have a
delayed implantation of up to three months.

TABLE 3
Production of anti-ZP antibodies by harbour seals
immunized with ZP in a single injection at three doses

- Anti-ZP titer
(expressed as a % of a standard serum)*

zp Time (weeks)
(~g) Animal
2 3
F - 5.9
F 1.2 0.4
F 0.7 2.7
F 4.8
F 0.9
F 1.8 6.5
F - 12.0
F 18.0
F 1.1 6.9
F 0.4
F 4.7
F 1.6 - 11.0
F = adult female
- = no sample

* A value of 100% indicates the serum being tested has
the same titer as a pooled standard serum from rabbits
rendered infertile by immunization with SIZP.

EXPERIMENT 4
Immunization of Grey Seals
Field studies of grey seals on Sable Island have
been conducted and are still in progress. Grey seals go to
Sable Island every year to have pups and breed and were
therefore used in order to study the immunocontraceptive
ability of the vaccine preparation. One group of female
grey seals (approximately 100 animals) was immunized during
the January 1992 breeding season with liposomes in FCA
SU~ ITE SHEET

~093/25Z31 ~1 3 7 3 6 3 12 PCT/CA93/0~239


(control) while the other group of 100 animals was
immunized with ZP in liposomes in FCA. In January 1993 the
seals that returned to Sable Island were tested for anti-ZP
titers. The results of this study are shown in Table 4.
As expected, the control group of seals had antibody titers
of zero. The experimental group, on the other hand, had
appreciable titers. Providing anti-ZP titers persist for
1 year, it is expected that females in the experiment group
will not conceive and therefore should not return to Sable
Island in 1994.
It should also be noted that fewer seals in the
experimental group returned to Sable Island during the 1993
season (51 of 85 expected) than the control group (76 of 85
expected). These results demonstrate that the liposome
vaccine was effective as an immunocontraceptive even in the
1992 breeding season, immediately following administration
of the vaccine. Anti-ZP antibodies inhibit implantation of
embryos in the uterus. As seals have delayed implantation
of about 3 months, antibody production had sufficient time
to build up to concentrations which inhibited implantation
(Henderson et al, 1988). This explanation is supported by
recapture of three seals (#44, #65 and #66) which had been
radio tracked. Their sera had anti-ZP titers of 12, 34 and
56%, respectively, 6 months after being immunized. The
seals that return to Sable Island in 1994 will be further
analyzed.




SVB~ 111 IJTE SHEET

W O 93/25231 ~ 1 3 7 3 6 3 PCT/CA93/00239

13
TABLE 4
Anti-ZP titer3 (a~ a % of a standard Yerum) in grey ~eal~
one year after injection with lipo~ome~/FCA (controls~
or ZP in lipo30me.~/FCA (experimental)
Control~ Bxperimental
Seal # Titer (%) Seal # Titer (%)
1 0 2 12
o 5 11
7 0 6 17
8 0 7 38
16 0 8 71
19 0 10 6
27 0 11 4
76 0 15 0
77 0 18 19
86 0 19 37
87 0 20 5
88 0 22 41
94 0 27 21
96 0 29 3
103 0 31 2
112 0 33 67
117 0 34 20
27
39 8
41 32
42 4
- 47 16
48 58
52 5
53 42
12
57 25
61 10
62 3
63 4
51
67 2
68 4
33
71 26
73 2
74 91
76 75
77 2
78 26
79 76
81 77
84 29
88 26
91 5
93 16
94 26
96 49
101 10
104 6
A value of 100% indicate~ the ~erum being tested ha~ the 3ame
titer as a pooled 3tandard serum from rabbit~ rendered infertile
by i iz~tion with SIZP.


SU~:~ 111 IJTE SHEET

WO93/25231 PCT/CA93/00239
'~13736~
14 _
EXPERIMENT 5
After the filing of the priority application
(USSN 07/892,807), the inventors cont_nued their research
to develop and improve the previously mentioned liposomal
vaccine formulations incorporating zona pellucida (ZP)
antigens. One new embodiment involved the incorporation of
a freeze-dried liposome containing ZP and adjuvant into a
BallistiVet~ biobullet. A biobullet is a gelatin capsule
adapted to incorporate antigens for the purpose of vaccin-
ating large animals. The biobullet is shot into the animalwith a specially designed gun thus avoiding the need to
restrain and inject the animals.
Procedure
1. Preparation of Biobullet
The biobullets were obtained from BallistiVet
(Minneapolis, MN) and packed with the appropriate vaccine
or control formulation as described below.
2. Immunization of Rabbits
Rabbits (two rabbits for each treatment) were
injected i.m. (a + b below) or a biobullet was surgically
implanted tc below) as follows: (a) ZP (20 ~g) was encap-
sulated in liposomes prepared according to U.S. patent
4,485,054 and the liposomes were suspended in a mixture of
saline (0.25 ml) and FCA (0.25 ml); (b) ZP (20 ~g) encap-
sulated in liposomes prepared as above and suspended in amixture of saline (0.25 ml) and FIA (0.25 ml) and (c) ZP
(20 ~g) encapsulated in liposomes prepared as above then
freeze-dried and the freeze-dried powder suspended in FCA
(0.25 ml). This suspension was packed into a biobullet
(BallistiVet) and implanted i.m. by surgery.
Results:
The results of this study are shown in Table 5.
Administration of ZP in liposomes with FCA produced high
anti-porcine ZP antibody titers within one-two months.
Replacing FCA with FIA, only marginally reduced anti-ZP
antibody production. Use of freeze-dried liposomes with

SUB~ l l l UTE SHEEl'

WO93/2~231 ~ 13 7 3 6 3 PCT/CA93/00239


FCA in a biobullet resulted in an increased and prolonged
production of anti-ZP antibodies.

TABLE 5
Effect of adjuvants on the production of anti-ZP antibodies
by rabbits immunized with ZP in liposomes or freeze-dried
liposomes in biobullets.

Titer (% of standard serum)
Delivery/adjuvant Time (months)
1 2 3 4 5
Liposome/FCA 120 131 103 24 16
38 38 24 17 19
Liposome/FIA 12 26 38 6
91 97 73 18 19
Biobullet/FCA 159 149 215 27 66
196 344 128 98

FCA = Freund's complete adjuvant.
FIA = Freund's incomplete adjuvant.

EXPERIMENT 6
In this experiment, the three seals from
Experiment 2 in groups (a) and (b) that were tested up
until month 22 for anti-ZP antibodies were given a booster
of SIZP ~90 ~g) in freeze-dried liposomes with Freund's
Incomplete Adjuvant (FIA) delivered with a BallistiVet
biobullet into the hindquarter of each seal. Serum samples
were taken after 4 weeks (month 23), 6 weeks (month 23.5),
8 weeks (month 24) and 12 weeks (month 25) after
administration of the booster and anti-porcine ZP titers
measured.
Results:
The results are shown in Table 6. A booster
administered using a biobullet increased the anti-ZP
antibodies to high levels which peaked 6-8 weeks later. It
is expected that at least one more serum sample will be
taken from these animals before they are released.

SUBS~ JTE SHEET

16
TABLE 6

Anti-ZP Titer (% of a standard serum)
Antigen Time (months)
23 23.5 24 25
ZP ~15 ~g) - _
FCA/lipos 116 159 119 79
ZP (90 ~g) 54 59 68 33
FCA/lipos 15 21 41 13

EXPERIMENT 7
The zone pellucida (ZP) antigen used in the pre-
viously mentioned seal studies was porcine derived.
Therefore in order to demons~rate that antibodies raised in
sea~s to porcine ZP do cross-react with seal oocytes, the
following experiment was conducted.
Procedure:
Antibodies from unimmunized harp seals and harp
seals immunized with porcine ZP were purified by affinity
chromatography using a Protein A column (Pierce Chemical
Co., U.S.A.). The two antibody preparations were labelled
(14C) by reductive methylation ~Jentoft, N. and D.G. Dear-
born, 1979, Labelling of proteins by reductive methylation
using sodium cyanoborohydride, J. Biol. Chem. 254: 4359-
4365).~ Labelled antibodies were incubated with either sealor porcine oocytes overnight at 2~C. Unbound antibc~y was
removed by centrifugation with washing (2X's) with saline.
Labelled antibody bound to oocytes was determined by liquid
scintillation counting (LKB racbeta* instrument). Oocytes
were counted using a microscope and a haemocytometer-l~ke
counting chamber.
Results:
The results are shown in Table 7. Seal and
porcine oocytes incubated wlth labelled antibodies from
control seals bound only small quantities of antibody
~25-52 DPM).
Seal oocytes (93) bound 14C-labelled anti-porcine
ZP antibodies, 119 DPM (35.8 ng protein/oocyte) whereas

*Trad e-mark
' " 74268-12 (S)


. .

WO93/25231 ~13 7 3 6 3 PCT/CA93/~239

17
porcine oocytes (96) bound more antibody (250 DPM; 72.9 ng
protein/oocyte). This result suggests that about one-half
the antibodies produced against porcine ZP in seals recog-
nized and bound to seal ZP. Increasing the number of seal
oocytes from 93 to 178 resulted in an increase in the quan-
tity of antibody bound to oocytes (from 119 DPM to 169 DPM)
demonstrating all sites on the seal oocytes capable of
binding antibody were occupied. Increasing the number of
porcine oocytes from 96 to 192, 432 and 864 oocytes
resulted in no significant increase in the quantity of
14C-labelled antibody bound (250, 228, 236 and 277 DPM).
This result demonstrated that 96 porcine oocytes bound all
anti-porcine-ZP antibody present in the serum from the
immunized seal.
These results verify that anti-porcine ZP anti-
bodies bind to seal oocytes and provide evidence that
immunocontraception may be explained by the binding of
antibodies to the oocyte surface.

TABLE 7
Binding of 14C-labelled anti-ZP antibodies
to pig and seal oocytes

oocytes Ab from control seal Ab from i ized
seal
source number Ab bound Ab protein Ab bound Ab protein
~DPM)bound (DPM) bound
(ng/oocyte) (ng/oocyte)
seal 178 38 5.0 18926.6
- seal 93 25 6.3 11935.8
porcine864 52 1.4 2779.0
porcine432 47 2.6 23615.3
porcine192 45 5.5 22833.3
porcine 96 55 13.6 25072.9
The Ab from the control seal added to the oocytes contained 2124 DPM
(specific activity, 42,309 DPM/mg protein) whereas the Ab from the
zed seal added to the oocytes contA;ned 1672 DPM (specific
activity, 35,685 DPM/mg protein).

SUMMARY
The above described experiments demonstrate that
incorporating a zona pellucida antigen with a suitable
adjuvant into a liposome delivery system results in an
Sl~ T

WO93/25231 ~ 1 3 7 3 6 3 PCT/CA93/00239

18
effective vaccine system for immunocontraception. The
studies have shown that a single injection of the vaccine
induces levels of anti-zona pellucida antibodies up to
22 months. A single injection vaccine is clearly
advantageous for any immunocontraceptive protocol.
Initial studies have shown that incorporating a
freeze-dried liposome containing the ZP antigen and
adjuvant into a biobullet also induces high levels of anti-
ZP antibodies in the seals. Such a delivery system is
advantageous since it does not require that the ~nlm~l S be
restrained to be injected with the vaccine.
While the above described experiments relate to
certain embodiments of the present invention, it is to be
appreciated that various modifications can be made to the
vaccine composition without departing from the scope and
spirit of the invention. For example, while the above
described examples relate to porcine ZP3, any suitable and
effective zona pellucida antigen, of porcine or other
origin, may be employed. In fact, studies have shown that
the genes that code for zona pellucida are conserved among
mammals. The zona pellucida antigen may also be purified
from oocytes or alternatively, a recombinant ZP antigen may
be used. Modifications can also be made to the liposome
delivery system. In particular, any liposomal system ~such
as freeze dried, liquid or semi-solid forms) which allows
for the slow, controlled release of the antigen is consid-
ered within the scope of the present invention. Finally,
the adjuvant used can also be modified, as long as it is
effective in enhancing the immune response to the vaccine
and is suitable for the intended application.




SUBS 111 LITE SHEET

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États administratifs

Titre Date
Date de délivrance prévu 1999-06-15
(86) Date de dépôt PCT 1993-06-07
(87) Date de publication PCT 1993-12-23
(85) Entrée nationale 1994-12-05
Requête d'examen 1997-11-03
(45) Délivré 1999-06-15
Réputé périmé 2012-06-07

Historique d'abandonnement

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Historique des paiements

Type de taxes Anniversaire Échéance Montant payé Date payée
Le dépôt d'une demande de brevet 0,00 $ 1994-12-05
Taxe de maintien en état - Demande - nouvelle loi 2 1995-06-07 100,00 $ 1995-01-25
Enregistrement de documents 0,00 $ 1995-06-22
Taxe de maintien en état - Demande - nouvelle loi 3 1996-06-07 100,00 $ 1996-05-29
Taxe de maintien en état - Demande - nouvelle loi 4 1997-06-09 100,00 $ 1997-05-20
Examen avancé 100,00 $ 1997-11-03
Requête d'examen 400,00 $ 1997-11-03
Taxe de maintien en état - Demande - nouvelle loi 5 1998-06-08 150,00 $ 1998-05-11
Taxe finale 300,00 $ 1999-03-17
Taxe de maintien en état - Demande - nouvelle loi 6 1999-06-07 150,00 $ 1999-04-23
Taxe de maintien en état - brevet - nouvelle loi 7 2000-06-07 75,00 $ 2000-04-20
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Taxe de maintien en état - brevet - nouvelle loi 9 2002-06-07 150,00 $ 2002-05-09
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Enregistrement de documents 100,00 $ 2007-02-14
Taxe de maintien en état - brevet - nouvelle loi 14 2007-06-07 250,00 $ 2007-05-25
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Titulaires au dossier

Les titulaires actuels et antérieures au dossier sont affichés en ordre alphabétique.

Titulaires actuels au dossier
IMMUNOVACCINE TECHNOLOGIES INC.
Titulaires antérieures au dossier
BROWN, ROBERT
DALHOUSIE UNIVERSITY
KIMMINS, WARWICK
MEZEI, MICHAEL
POHAJDAK, BILL
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Description 1998-09-09 19 725
Revendications 1998-09-09 2 35
Page couverture 1995-08-14 1 19
Abrégé 1993-12-23 1 45
Description 1993-12-23 18 706
Revendications 1993-12-23 2 37
Description 1998-03-31 19 704
Revendications 1998-03-31 2 32
Page couverture 1999-06-11 1 27
Correspondance 1999-03-17 1 39
Poursuite-Amendment 1998-03-31 19 594
Poursuite-Amendment 1997-12-02 2 58
Cession 1994-12-05 8 317
PCT 1994-12-05 17 487
Poursuite-Amendment 1997-11-03 2 64
Correspondance 1995-07-20 5 114
Correspondance 2000-04-20 1 27
Poursuite-Amendment 1997-11-18 1 1
Taxes 2001-05-07 1 38
Correspondance 2001-05-07 1 34
Correspondance de la poursuite 1994-12-05 10 435
Cession 2007-02-14 4 105
Taxes 2007-05-25 1 37
Taxes 2008-05-28 1 36
Taxes 1997-05-20 1 41
Taxes 1996-05-29 1 47
Taxes 1995-01-25 1 53