Evaluation of immunogenicity and contraceptive efficacy of gamete-specific recombinant proteins for the development of contraceptive vaccine

Abstract

There is an urgent need for the management of the population of stray dogs as they act as a carrier of the deadly rabies virus. Contraceptive vaccines based on either spermatozoa or egg-specific proteins aiming to interfere in sperm–egg interactions have been proposed as one of the strategies for controlling the population of various animal species. To this end, a fusion protein TT-KK-ZP3; encompassing promiscuous T cell epitope of tetanus toxoid (TT), adilysine linker (KK)anddogzonapellucidaglycoprotein-3 (ZP3)without any affinity tag has been expressed and purified from E. coli. Production of the recombinant protein has been successfully up-scaled at fermenter level. Refolded TT-KK-ZP3 showed the presence of the defined secondary structure in CD and fluorescent spectroscopy. Female FvB/J mice immunized with TT-KK-ZP3 showed a dosedependent increase in antibody titre that correlated well with reduction in fertility. Antibodies against TT-KK-ZP3 showed binding with mouse and dog ZP matrix in indirect immunofluorescence. Additionally, a chimeric polypeptide comprising TT, KK, C-terminus fragment of ZP domain of dog ZP3 (ZP3c-term), a triglycine spacer (GGG) and a fragment of dog spermatozoa-specific protein, Izumo (Iz), was expressed and purified from E. coli (TT-KK-ZP3-GGG-Iz) without any affinity tag. Female mice immunized with TTKK-ZP3-GGG-Iz showed specific antibody response against ZP3c-term as well as Izumo in ELISA. However, antibody titre against Izumo was higher compared with ZP3c-term. Antibodies reacted with mouse and dog ZP matrix and acrosome reacted mouse and dog spermatozoa. A physical mixture of individually expressed ZP3c-term and Izumo also elicited a similar kind of immune response, but antibody titre was low compared with chimeric protein. Groups of mice were also immunized with Izumo and ZP3c-term individually. All the groups showed varying degrees of curtailment in fertility compared with the controls. Mouse in vitro fertilization was inhibited significantly in the presence of immune serum from mice immunized with any of the above recombinant proteins. It is likely that these endeavours might help us to propose a candidate vaccine for the management of the population of the street dogs that may eventually lower the burden of rabies infection.