FERTILITY IMPAIRING VACCINE and METHOD of USE

Detailed Description of the Preferred Embodiments The fertility impairing vaccine of the invention comprises an antigen comprising zona pellucida glycoprotein, preferably substantially pure zona pellucida glycoprotein, or an antigenic fragment thereof. Preferably, zona pellucida glycoprotein is a total porcine zona pellucida glycoprotein. A total zona pellucida glycoprotein preparation obtained from pig ovaries includes all three major heavily glycosylated porcine zona pellucida glycoproteins: pZPI, pZP3a and pZP3,. pZP3a and pZP3 P each have reported molecular weights of about 55 kD, and pZP1 has a reported molecular weight of about 82 kD. The amino acid sequences of these three glycoproteins are known (J. D. Harris et al., DNA Seq., 4,361-393 (1994)). Other reported pZP glycoproteins are believed to be degradation products of pZP 1.

Purity of the zona pellucida glycoprotein can be evaluated analytically using a combination or series of two-dimensional sodium dodecyl sulfate polyacrylamide gels (SDS-polyacrylamide gel electrophoresis, or SDS- PAGE) with silver staining, Coomassie Blue staining, and Western blot analysis, as described in the following Examples. Glycoproteins typically migrate electrophoretically in gels as broad smears rather than narrow bands, as a result of the variable levels of negative charge present in the constituent oligosaccharide chains. A"substantially pure"total zona pellucida glycoprotein preparation isolated from pig ovaries migrates as two distinct smears in the gel electrophoretic experiments (one smaller smear representing pZP 1, and one larger smear representing pZP3a and pZP3p), and shows immunological reactivity in Western blot analysis using a polyclonal antibody raised in rabbits to highly purified total porcine zona pellucida glycoprotein. In a substantially pure zona pellucida glycoprotein preparation used for fertility impairment, there are no detectable contaminating proteins. The absence of detectable contaminating proteins is determined by demonstrating that there are no proteins in the preparation that have electromigration patterns different from those exhibited by the zona pellucida glycoproteins as determined by two-dimensional SDS-PAGE (silver-stained) or Western blot analyses of two-dimensional SDS- PAGE gels. An antigenic fragment of a zona pellucida glycoprotein is a peptide fragment, preferably a glycosylated peptide fragment, that elicits an immune response characterized by detectable anti-pZP antibody levels in the subject using ELISA as described in Example II. The peptide fragment preferably contains more than seven amino acids, more preferably at least about 10 amino acids, most preferably at least about 20 amino acids.

The zona pellucida glycoprotein used in the present vaccine is preferably a naturally occurring glycoprotein or a chemically or enzymatically synthesized glycoprotein. The glycoprotein is preferably not a recombinant glycoprotein, but use of a recombinant glycoprotein in the present vaccine is not necessarily excluded in alternative embodiments of the invention.

The vaccine of the invention preferably additionally includes an immunological adjuvant to enhance the immunological response of the subject to the glycoprotein antigen. Examples of adjuvants include Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, and an adjuvant comprising an immunostimulant such as synthetic trehalose dicorynomycolate (STDCM) and an oil such as squalene oil (see P. Willis et al., J. Equine Vet. Sci., 14,364-370 (1994)). An adjuvant comprising synthetic trehalose dicorynemycolate, squalene oil, and a surfactant such as lecithin is preferred. Lecithin typically includes phosphatidyl choline.

In a preferred embodiment the vaccine comprises oil, preferably a biodegradable oil such as squalene oil, in an amount of about 2.5% to about 15%, preferably about 8% to about 12%. In preparing the vaccine it is advantageous to combine a concentrated oily adjuvant composition with an aqueous solution of the antigen, pZP glycoprotein. Typically, the vaccine is prepared using an adjuvant concentrate which contains lecithin (about 5% to about 15 % wt/vol, preferably about 12% wt/vol) and STDCM (preferably about 25 mg/mL to about 50 mg/mL) in squalene oil. The term % wt/vol means grams per 100 mL of liquid. The aqueous solution containing the isolated pZP glycoprotein is typically a phosphate-buffered saline (PBS) solution, and additionally preferably contains Tween 80 (about 0.2% vol/vol to about 0.8% vol/vol, preferably about 0.4% vol/vol). See J. A. Rudbach et al.,"Ribi Adjuvants: Chemistry, Biology and Utility in Vaccines for Human and Veterinary Medicine,"in The Theorv and Practical Application of Adjuvants, D. E. S. Stewart-Tull, Ed., John Wiley & Sons, New York, NY (1995)).

Homogenization of the oily adjuvant concentrate with the aqueous pZP solution can be accomplished using any convenient means known in the art, such that the oil disperses within the aqueous solution to form an oil in water emulsion. Oil droplet sizes of about 200 nm or less are particularly preferred as they produce a more uniform and stable suspension. A particularly preferred vaccine comprises predetermined amounts of pZP and STDCM in an emulsion containing about 10% squalene oil and about 90% aqueous phase.

The invention further includes a method for administering a fertility impairing vaccine as described herein in a manner effective to cause impaired fertility in an animal, preferably a carnivore (i. e., a member of the order Carnivora). Preferably the carnivore is not a primate, and is a dog or a cat, more preferably a dog. Impairment of fertility in an animal in accordance with the invention can take the form of either immunocontraception and immunosteriliztion. Immunosterilization means permanent, irreversible infertility, in contrast to immunocontraception wherein infertility is temporary or transient, and reversible. Immunocontraception and immunosterilization are both dependent on the antibody titer level in the serum of the subject, but immunosterilization is typically the result of ovarian pathology caused by vaccine administration and high titers of anti-pZP antibodies, as evidenced by, for example, total destruction of the zona pellucida glycoproteins and/or influx of leukocytes into the follicles. Reducing the number of boosters leads to lower antibody titers which results in immunocontraception (i. e., infertility that is temporary and reversible) instead of immunosterilization.

The vaccine is administered in a manner and an amount effective to cause the desired infertility in the mammalian subject.

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