Adenoviral Vectored Gonadotropin Releasing Hormone Vaccine for Estrus Suppression in Mares

Abstract

The objective of this study was to evaluate an adenoviral vectored gonadotropin releasing hormone (GnRH) vaccine as a method to suppress estrus behavior and cyclicity in mares. An additional objective was to determine the effects of a heterologous vaccination strategy against gonadotropin releasing hormone on estrus behavior and cyclicity in mares using an adenoviral vectored gonadotropin releasing hormone vaccine to prime, and a protein based gonadotropin releasing hormone vaccine to boost. Twelve normal cyclic mares were included in the study which was divided into two phases. The first phase (weeks 0-46) included one ovulatory season and phase two (weeks 47-70) included the subsequent ovulatory season. During phase one, treatment mares (n=5) were vaccinated twice, 4 weeks apart, with an adenoviral vectored gonadotropin releasing hormone vaccine. The vaccine was a replication-defective E1/E3 deleted adenovirus (Ad5) vector expressing antigens consisting of 16 multimers of GnRH, bacterial leukotoxin, T-helper epitopes, and other various hinge and linker amino acids (Ad-GnRH). Each 1 milliliter dose of the vaccine contained 4.64E10 infectious units. Five additional mares served as controls for estrus behavior, cyclicity, and seasonality. During phase two, mares that had been vaccinated during phase one (previous ovulatory season) were administered a single vaccination using a quarter of the labeled dose (sub-effective) of a protein based gonadotropin releasing hormone vaccine (100 µg protein conjugate per quarter of the labeled dose: Equity® Oestrous Control Vaccine, Zoetis, Australia). Two naïve mares (protein vaccine control mares) received an equivalent dose of the protein based gonadotropin releasing hormone vaccine to determine if the 100 µg protein conjugate dose was sub-effective for suppression of cyclicity and estrus. Anti-GnRH antibodies, estrus behavior, reproductive tract sonography, and serum progesterone concentrations were monitored over two consecutive breeding seasons. Following homologous prime and boost using Ad-GnRH, all treatment mares developed anti-GnRH antibodies and the antibody response remained significantly different from that of time zero for 32 weeks during phase one. There was no effect on mean interestrus interval, reproductive cyclicity, or estrus behavior. Following heterologous boost during phase two, all treatment mares experienced an anti-GnRH antibody response that was maintained for at least 17 weeks (remainder of study period). All treatment mares became anestrus based on serum progesterone concentrations and transrectal sonographic findings. Estrus behavior became erratic and unpredictable, and therefore, interestrus interval could not be calculated. Protein vaccine control mares developed anti-GnRH antibodies after vaccination and the response was maintained for 15 weeks. There was no effect on interestrus interval, reproductive cyclicity, or estrus behavior. The day following Ad-GnRH boost, three of five treatment mares developed a non-painful 3-5cm raised nodule at the injection site. Following heterologous boost, two of four treatment mares developed a small (<2cm) raised, non-painful nodule. All injection sites reactions resolved within three days without treatment. This study demonstrates that mares are capable of developing an anti-GnRH antibody response to homologous immunization using a replication-defective E1/E3 deleted replication-defective adenovirus vector encoding GnRH peptide, bacterial leukotoxin, and T-helper epitopes. Homologues prime-boost vaccination of mares with Ad-GnRH at the dose and frequency used in this study does not result in suppression of reproductive cyclicity and estrus behavior. This study demonstrates that heterologous prime-boost vaccination of mares using an Ad-GnRH prime and protein based GnRH vaccine boost results in an antibody response that suppresses reproductive cyclicity, and interferes with estrus behavior. Vaccine-induced suppression of reproductive cyclicity and estrus was maintained for at least 17 weeks. Vaccinated mares experience minimal side effects following vaccination with either Ad-GnRH or heterologous boost using a sub-effective dose (100µg protein conjugate) of a protein based GnRH vaccine.