A number of proteins in the hypothalamic-pituitary-gonadal axis have been targeted for immunocontraception in other species. Zona pellucida (ZP), the glycoprotein matrix surrounding the mammalian egg, regulates sperm–egg interactions during fertilization and is subsequently removed prior to implantation.28–30 The relatively high amino acid sequence homology across species of some portions of the protein suggests that immunocontraceptive vaccines based on ZP proteins of one species may induce adequate cross-reactivity to immunize other species.31,32 Indeed, native porcine ZP (pZP) isolated from pig ovaries collected at abattoirs has been used for successful long-term contraception in horses, deer, rabbits, elephants, seals, and other species.19,28,33 In most reports, successful ZP immunocontraception is not associated with ovarian disruption leading to the suppression of estrous cycling, which could be considered a weakness of this approach. Cats are seasonally polyestrous, and failure to conceive after breeding triggers a period of pseudopregnancy followed by return to estrus. In the long term, this may lead to an increase in complaints about nuisance behaviors associated with the cat breeding season and uterine or mammary diseases in vaccinated cats.
In the first report investigating pZP for immunocontraception in cats, a vaccine containing pZP and Freund’s adjuvant [Freund’s complete adjuvant with killed mycobacteria in oil (FCA) for the first vaccine dose and incomplete adjuvant lacking mycobacteria (FIA) for subsequent doses] was administered subcutaneously to eight adult cats five times over a period of 92–150 days.34 Serum ZP antibodies reacted more strongly against pig oocytes than against cat oocytes. In a subsequent 3-month breeding trial in five of the cats, only one became pregnant, suggesting that pZP may have immunocontraceptive potential in female cats. A subsequent study evaluating the binding activity of rabbit anti-pZP antibodies in immunohistochemical (IHC) evaluation of cat ovaries identified cross-reactivity with cat oocytes, further supporting this potential.35 In contrast, another study failed to confirm cross-reactivity of rabbit anti-pZP antibodies with feline ZP (fZP).36 Antibodies against pZP failed to block sperm binding or in vitro fertilization of cat oocytes, whereas antibodies raised in rabbits against fZP did inhibit fertilization. IHC studies demonstrated that pZP antibodies did not bind to cat oocytes, but fZP antibodies did. In a third study, antibodies raised in rats against a portion of the fZP protein containing an epitope recognized by rabbit anti-fZP serum (ZPB amino acid residue 130–149) reduced in vitro sperm binding and fertilization of feline oocytes.37 These studies are contradictory, clouding the issue of whether ZP has contraceptive potential in cats. In addition, findings in all three studies were based on antibodies produced in rabbits or rats, not in cats, and none was confirmed in breeding trials. A fourth study comparing the effect of vaccines based on subunits of pZP confirmed that three monthly doses of pZPB+C emulsified in FCA (first vaccine) or FIA (second and third vaccines) and injected in multiple locations subcutaneously induced antibodies in 2–3-year-old cats that failed to bind to cat oocytes during IHC or to prevent pregnancy.29 The investigators also tested DNA vaccines containing sequences coding for fZPA or fZPB+C in which three doses were administered intramuscularly at monthly intervals. In a breeding trial spanning a single estrous cycle, a lower proportion of cats receiving the DNA vaccines became pregnant than controls. However, a low conception rate in the controls coupled with a low number of cats that attempted to breed rendered the differences statistically insignificant. Histologic findings suggestive of possible underlying reproductive pathology in some cats contributed to further uncertainty regarding the effect of fZP vaccination on fertility of cats. Contradictory or ambiguous findings may be explained by a lack of standardization among studies, including differences in the source and preparation of ZP used for immunization, use of different adjuvants, variable vaccination schedules and procedures, and use of surrogate outcome markers in place of breeding trials in the target species.
In the first study in cats using a pZP vaccine with a history of multiyear immunocontraception in other species, three groups of 10 8- to 12-week-old kittens were vaccinated once intramuscularly with one of two vaccine formulations or with a sham vaccine lacking pZP.30 The vaccine, SpayVac™ (Immunovaccine Technologies, Halifax, NS, Canada), was composed of soluble pZP isolated from pig ovaries collected at abattoirs encapsulated in multilamellar liposomes for slow release and emulsified with one of two adjuvants, FCA or alum with oil and mannide oleate. Cats responded with the rapid induction of anti-pZP antibodies, but similar to previous studies, IHC revealed binding only to pig oocytes and not cat oocytes. All cats maintained normal estrous cycling and became pregnant. There were no differences in time to pregnancy or litter size among the three treatment groups. Ovaries removed from cats after parturition failed to show any binding of antibodies to oocytes in treated cats. This study confirmed earlier concerns questioning the degree of cross-reactivity between pZP and fZP.
It remained to be determined whether the ZP of species other than the pig would share more effective antigenic determinants with feline ZP versus whether the cat was unique in its failure to respond immunologically to this self-antigen. It has been shown that homology in ZP sequences is greatest within the same mammalian class, suggesting that ZP from species more closely related to the cat may be more suitable candidates.31,32 In addition, the previous finding that antibodies raised in rabbits against fZP bound to cat oocytes suggested that ZP isolated from cat ovaries might be capable of stimulating the production of antibodies that recognize fZP. To explore the effect of alternative sources of ZP, native soluble ZP isolated from the ovaries of ferrets, mink, dogs, cows, and cats were used in SpayVac™ formulated with FCA.38 Groups of three 15- to 20-week-old kittens were vaccinated once intramuscularly with SpayVac™ containing one of the five species ZP. As previously shown for pZP, anti-ZP antibody titers were generally higher against the species ZP contained in the vaccine than against fZP. None of the sera reacted with cat oocytes, and all cats became pregnant. Interestingly, the three cats vaccinated against fZP had litter sizes of one, one, and five, respectively. This suggested that immunization with fZP may reduce reproductive success. To further evaluate this observation, these three cats were boostered with fZP emulsified in FIA 32 weeks after the first dose. A second round of breeding beginning 5 weeks after the booster resulted in litter sizes of four, six, and four (stillborn), respectively, reducing enthusiasm for the potential of fZP as a contraceptive antigen in cats.
The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) controls pituitary release of LH and FSH, which in turn control testosterone and spermatogenesis in the male and estrogen and follicular development in the female.39,40 As this master reproductive hormone controls fertility and behavioral responses in both sexes, it is an ideal target antigen for immunocontraception.21,33 GnRH is widely conserved across all mammalian species. This is both an opportunity to develop immunocontraceptives with activity in multiple overabundant species and a threat in regard to safeguarding non-target species and personnel who must handle the products. As a small self-antigen, GnRH is a poor immunogen. Immune recognition is enhanced by conjugating GnRH to large foreign proteins and administration in the presence of immune-stimulating adjuvants.
In the first report in which the hypothalamic-pituitary-gonadal axis was targeted for fertility control in cats, synthetic GnRH was conjugated to tetanus toxoid, emulsified, and mixed with N-acytyl-nor-muramyl-l-d-isoglutamine as an adjuvant.41 Six cats vaccinated three times at weeks 1, 2, and 4 experienced only modest and transient decreases in serum testosterone, whereas dogs vaccinated with a similar protocol experienced several months of testosterone levels consistent with immunocastration before recovering fertility. In a second study, an attempt to block the downstream effects of GnRH stimulation by prevention of LH binding utilized vaccination against LH receptors. Bovine LH receptors emulsified in N-acetylglucosaminyl-(β1-4) N-acetylmuramyl-l-alanyl-d-isoglutamine adjuvant prepared from mycobacterium cell walls were packaged into silastic implants. Implants were placed via surgical incision in the interscapular space in seven anesthetized 8-month-old female cats.42 Cats were also vaccinated intramuscularly with the same preparation on days 98, 139, 160, and 193. Cats produced antibodies against LH, resulting in the suppression of serum progesterone for more than a year. On day 345, cats were stimulated with GnRH, resulting in a rise in serum LH indicating that pituitary function remained intact. Breeding studies were not performed. In a third study, a recombinant fusion protein containing multiple copies of GnRH fused to a carrier protein fragment of leukotoxin A was emulsified in an oil-in-water adjuvant containing the immunostimulant dimethyl dioctadecyl ammonium bromide and administered subcutaneously at eight and twelve weeks of age to ten female and four male kittens.43 Tissue reactions were palpable at the injection site in all cats, but resolved in most cats within 28 days. Immunocontraceptive GnRH antibody titers persisted past 20 months in 14 of 15 cats. In males, this was associated with undetectable serum testosterone concentrations, whereas in females, it was reflected in low progesterone concentrations and failure to conceive in a breeding trial. Booster vaccines given on day 643 resulted in even higher GnRH antibody titers.
The first description of successful immunization against a self-antigen in cats following a single vaccination occurred when 9–12-month-old male cats were vaccinated intramuscularly with GonaCon™ (Fort Collins, CO, USA).44 The GonaCon™ vaccine is constructed with multiple copies of synthetic GnRH coupled to keyhole limpet hemocyanin carrier protein and emulsified in an oil and water adjuvant containing killed mycobacterium (AdjuVac™; Fort Dodge Animal Health, Fort Dodge, IA, USA). Six of nine vaccinated cats responded with persistently high GnRH antibody titers, which correlated with undetectable serum testosterone concentrations by 3 months post-injection. Immunocastration was associated with progressive decrease in scrotal volume and regression of androgen-dependent penile spines (Fig. 1). Semen analysis performed 6 months post-vaccination demonstrated absence of motile sperm in the responding cats. Testicular weights in those cats were only one quarter that of the sham-treated cats. Three cats with poor response to GnRH vaccination as defined by low GnRH antibody titers had testosterone concentrations, sperm counts, and scrotal volumes that were intermediate between the responding cats and the sham-treated cats.
Figure 1. Phenotypic responses of cats 6 months after receiving a single sham treatment (left) or GonaCon™, a vaccine containing synthetic GnRH coupled to keyhole limpet hemocyanin and emulsified in oil and killed mycobacterium (box). Three cats receiving a sham treatment had normal-sized testes (a), whereas cats vaccinated against GnRH had variable testicular atrophy depending on the GnRH titer and resulting suppression of testosterone (b). Androgen-dependant penile spines remained prominent in sham-treated cats (c) and regressed in cats that responded to GnRH vaccination (d).
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Successful immunocastration of male cats with a single GnRH vaccination in the short-term study was followed by a multiyear study in which fertility of GonaCon™-vaccinated male cats was tested in a breeding trial.45 For the nine of 12 vaccinated cats that responded to vaccination with high GnRH antibody titers, the median onset of testosterone becoming undetectable was 2 months (range 1–12 months) and the median duration of effect was 14 months (range 5–33 months) (Fig. 2). One cat still had undetectable testosterone at the end of the observation period 34 months after treatment. Loss of detectable testosterone was generally followed in 1–2 months by azoospermia, and restoration of normal sperm counts lagged behind recovery of testosterone by 2 months (Fig. 3). Semen characteristics, including morphology and viability, were similar in cats prior to treatment and following recovery of fertility. Three treated cats that failed to respond with high GnRH antibody titers had minimal to no suppression of testosterone. The average time from introduction of the female cats to successful breeding was 12 months (range 3–12 months) for the responding cats, 5 months (range 5–6 months) for the poorly responding cats, and 3 months (range 0–9 months) for the sham-treated cats. In one extreme case, GnRH antibody titer did not begin to increase until 6 months post-vaccination, testosterone was not suppressed until 12 months, and azoospermia did not occur until 14 months. In this cat, the contraceptive effect lasted 14 months, after which GnRH antibody titer waned, normal testosterone concentration and semen characteristics recovered, and the cat sired a litter.46 The mechanism for this delayed response is unknown. A second late peak of response to GonaCon™ after initial decline in GnRH antibodies has been reported in rats 8 months after vaccination47 and in a dog 6 months after vaccination,48 indicating persistence of antigen for prolonged periods of time in some individuals. Median litter size sired by the cats was not significantly different between the groups. In this study, GnRH immunocontraception showed efficacy in 75% of male cats following a single treatment, but response was highly variable and all but one cat recovered fertility by 3 years post-treatment.
Figure 2. Intervals during which testosterone was undetectable in individual male cats following a single dose of the GnRH vaccine GonaCon™. Onset and duration of immunocastration varied among cats, and three cats with a poor antibody response to vaccination never had more than 1 month without detectable serum testosterone.
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Figure 3. GnRH antibody titer correlation with testosterone (a), total sperm count (b), and scrotal volume (c) in a representative cat following a single dose of the GnRH vaccine GonaCon™. The GnRH antibody titer reached a contraceptive level within 1 month of vaccination after which testosterone and sperm count decreased to undetectable levels and scrotal volume decreased. GnRH antibody titer waned 12 months after vaccination, which was associated with recovery of testosterone production, normal sperm counts, and increased scrotal volume.
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Although suppression of sex hormones in male cats is valuable because of a reduction in nuisance behavior, population control is primarily dependent on fertility control in females. A breeding trial in female cats demonstrated multiyear immunocontraception in 11 of 15 8- to 14-month-old female cats vaccinated intramuscularly with a single dose of GonaCon™, including four cats still infertile 5 years after vaccination (Fig. 4).49 Similar to male cats, four females (27%) failed to produce high GnRH antibody titers and became pregnant <2 years after vaccination. Altogether, vaccinated cats had a significantly longer time to conception (median 39.7 months) compared with sham-treated cats (4.4 months). Late-onset injection site reactions developed in five of the cats. Histology revealed granulomatous inflammation containing inactivated mycobacteria from the adjuvant, suggesting that continued inflammation stimulated by the vaccine may be responsible for the durable immune response observed in a high proportion of vaccinated female cats. The duration of response to GonaCon™ was longer in female cats than in male cats. Less predictable immunocontraception of males compared with females has been observed in other species as well, possibly related to more difficulty in blocking continuously secreted GnRH in males compared with episodic secretion in females.50,51
Figure 4. Results of a breeding trial in female cats following a single sham treatment (n = 5 cats) or the GnRH vaccine GonaCon™ (n = 15 cats). Cats were group-housed with fertile males beginning 4 months after GnRH vaccination and remained until they were successfully bred. Four cats maintained high GnRH antibody titers and remained infertile throughout the study until it was completed 5 years following a single GnRH vaccination.
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