The immunogenicity of recombinant and dimeric gonadotrophin-releasing hormone vaccines incorporating a T-helper epitope and GnRH or repeated GnRH units

Abstract

In this study, we designed two linear peptides, GnRH–hinge–MVP, which consists of human gonadotrophin-releasing hormone (GnRH), hinge fragment 225–232/225′–232′ of human IgG1 and a T helper peptide from measles virus protein (MVP), and GnRH3–hinge–MVP, which contains three copies of GnRH (so termed GnRH3). The DNA constructs encoding for the two peptides were fused to the C-terminal encoding sequence of asparaginase, encompassing residues 199–326, through an acid-labile aspartyl–prolyl linker. The chimeric genes were expressed at high levels in Escherichia coli. The fusion proteins were purified to approximate homogeneity by means of washing the inclusion bodies and by ethanol precipitation. The GnRH–hinge–MVP or the GnRH3–hinge–MVP was released from the fusion proteins by cleavage with hydrochloric acid and further oxidized into double-chain miniproteins after purification. Both dimeric constructs proved to be efficient immunogens. It was shown that rats immunized with the immunogens generated antibodies specific for GnRH. The dimeric GnRH3–hinge–MVP containing three copies of GnRH in each chain induced a higher titre of anti-GnRH antibodies than the GnRH–hinge–MVP, containing a single copy of GnRH in each chain. These results demonstrate that combining multicopies or single copies of peptide with hinge fragment of human IgG and T helper peptide from measles virus protein can induce anti-peptide immune responses. Our data also suggest that these methods of preparation and dimerization of the recombinant polypeptides may provide a useful strategy for other polypeptide vaccine developments.