Title: Method to prevent fertilization in mammals by
administering a single dose of zona pellucida derived antigens, liposome
United States Patent: RE37,224
Inventors: Brown; Robert (Dartmouth, CA); Mezei; Michael
(late of Halifax, CA); Mezei; Catherine (Vancouver, CA); Pohajdak; Bill
(Dartmouth, CA); Kimmins; Warwick Charles (Halifax, CA)
Assignee: Dalhousie University (CA)
Appl. No.: 156159
Filed: July 23, 1998
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 in seals. Thus, the
vaccine according to the present invention is effective after a single
dose and is therefore very useful in immunocontraceptive protocols.
SUMMARY OF THE INVENTION
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 vaccine composition for the
immunocontraception of a mammal which comprises a zona pellucida derived
antigen incorporated into a liposome system.
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
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 .Iadd.parenterally .Iaddend.administering
an effective amount of the above-described vaccine.
DETAILED DESCRIPTION OF THE INVENTION
Isolation and purification of zona pellucide glycoproteins
Zona pellucidaglycoproteins 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 .mu.m). The oocytes were homogenized with
a glass-teflon apparatus and the homogenate was passed through a 40 um
screen to collect the fractured zonae. The fractured zonae were washed
with buffer and recollected on the 40 um screen. Zona pellucida
glycoproteins were solubilized by incubating the zonae in a water bath (73oC.
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 mealis zona pellucida. ZP3 is
the major macromolecular 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
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. Dunbar. 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
Claim 1 of 26 Claims
We claim: .[.
1. A method of preventing fertilization in a mammal for at least twelve
months which method consists of a single parenteral administration of an
effective amount of a vaccine composition comprising a zona pellucida
derived antigen and a Freund's adjuvant in a liposome formulation..]..[.
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