Abstract：
To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs.
Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs.
Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control.
These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.
Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs.
Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control.
These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.
摘要：
管理狗的数量（犬），已在雌性比格犬中评估了基于重组蛋白的避孕疫苗抑制生育能力的功效。
用大肠杆菌表达的重组猪ZP3与破伤风类毒素的混杂T细胞表位（TT-KK-pZP3）和猪ZP4与牛RNase的混杂T细胞表位的物理混合物免疫雌性比格犬（n=4），或与包含狗ZP3片段和两个具有适当混杂T细胞表位的GnRH拷贝的融合蛋白（dZP3-GnRH2）;对照动物仅接受明矾，佐剂。通过ELISA跟踪免疫动物的抗体滴度以及与雄性狗交配后的生育力状态。
每隔4周注射三次后，用TT-KK-pZP3+bRNase-KK-pZP4和dZP3-GnRH2的物理混合物对雌性狗进行主动免疫，导致产生针对相应重组蛋白的显著抗体滴度。用dZP3-GnRH2的主动免疫还导致产生与dZP3和GnRH反应的抗体。第383天的加强剂量导致抗体滴度增加,并且在第528天可以观察到针对相应重组蛋白的循环抗体。如通过间接免疫荧光测定法评估的,来自用TT-KK-pZP3+bRNase-KK-pZP4或dZP3-GnRH2免疫的狗的免疫血清样品中的抗体与天然犬ZP反应。交配研究表明，与佐剂对照相比，用dZP3-GnRH2免疫的雌性狗的怀孕次数减少，出生的幼崽数量显着减少。dZP3-GnRH2免疫组的妊娠减少与针对dZP3-GnRH2的抗体滴度相关。然而,与佐剂对照相比，用重组TT-KK-pZP3bRNase-KK-pZP4免疫并没有显着减少出生的幼崽数量。
这些研究揭示了基于重组dZP3-GnRH2的避孕疫苗降低雌性狗生育能力的潜力。因此，有必要进行大规模研究，以确定该重组蛋白用于社区犬种群管理的功效和安全性。
用大肠杆菌表达的重组猪ZP3与破伤风类毒素的混杂T细胞表位（TT-KK-pZP3）和猪ZP4与牛RNase的混杂T细胞表位的物理混合物免疫雌性比格犬（n=4），或与包含狗ZP3片段和两个具有适当混杂T细胞表位的GnRH拷贝的融合蛋白（dZP3-GnRH2）;对照动物仅接受明矾，佐剂。通过ELISA跟踪免疫动物的抗体滴度以及与雄性狗交配后的生育力状态。
每隔4周注射三次后，用TT-KK-pZP3+bRNase-KK-pZP4和dZP3-GnRH2的物理混合物对雌性狗进行主动免疫，导致产生针对相应重组蛋白的显著抗体滴度。用dZP3-GnRH2的主动免疫还导致产生与dZP3和GnRH反应的抗体。第383天的加强剂量导致抗体滴度增加,并且在第528天可以观察到针对相应重组蛋白的循环抗体。如通过间接免疫荧光测定法评估的,来自用TT-KK-pZP3+bRNase-KK-pZP4或dZP3-GnRH2免疫的狗的免疫血清样品中的抗体与天然犬ZP反应。交配研究表明，与佐剂对照相比，用dZP3-GnRH2免疫的雌性狗的怀孕次数减少，出生的幼崽数量显着减少。dZP3-GnRH2免疫组的妊娠减少与针对dZP3-GnRH2的抗体滴度相关。然而,与佐剂对照相比，用重组TT-KK-pZP3bRNase-KK-pZP4免疫并没有显着减少出生的幼崽数量。
这些研究揭示了基于重组dZP3-GnRH2的避孕疫苗降低雌性狗生育能力的潜力。因此，有必要进行大规模研究，以确定该重组蛋白用于社区犬种群管理的功效和安全性。
