DNA vaccine encoding chimeric protein encompassing epitopes of human ZP3 and ZP4: immunogenicity and characterization of antibodies

Abstract

Immunization with zona pellucida (ZP) glycoproteins leads to curtailment of fertility often associated with ovarian dysfunction. To avoid ovarian dysfunction, synthetic peptides corresponding to ZP glycoproteins have been proposed as candidate immunogens. In the present study, plasmid DNA encoding a human ZP glycoprotein-3 (ZP3) epitope corresponding to amino acid (aa) residues 334-343 and a human ZP glycoprotein-4 (ZP4) epitope corresponding to aa residues 251-273 separated by a triglycine spacer was constructed using the mammalian expression vector, VR1020. The plasmid DNA construct expressed both human ZP3 and ZP4 epitopes, as revealed by transient transfection of COS-1 (African green monkey, kidney) mammalian cells. Active immunization of female BALB/cJ mice with the DNA vaccine led to generation of antibodies reactive with baculovirus-expressed recombinant human ZP3, ZP4 and ZP3_(334-343aa)-GGG-ZP4_(251-273aa) synthetic peptide in an ELISA as well as T cell responses. Antibodies generated by the DNA vaccine also recognized native ZP. The immune sera significantly inhibited (p/</0.005) the binding of FITC-labeled ZP3 to capacitated human sperm, whereas no inhibition in the binding of FITC-labeled ZP4 was observed. However, a significant decrease in acrosomal exocytosis mediated by both recombinant human ZP3 (p/</0.005) and ZP4 (p/</0.005) was observed in presence of the immune sera. These studies demonstrate that a DNA vaccine can be designed to elicit antibodies against small epitopes of ZP.