Active immunisation of mice with GnRH lipopeptide vaccine candidates: Importance of T helper or multi-dimer GnRH epitope Daryn Goodwin a , Pavla Simerska a , Cheng-Hung Chang a , Friederike M. Mansfeld a , Pegah Varamini a , Michael J. D’Occhio b , Istvan Toth a,c,⇑ a The School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia 4072, Queensland, Australia b The Faculty of Agriculture and Environment, The University Sydney, Camden 2570, New South Wales, Australia c The School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of Queensland, Woolloongabba 4102, Queensland, Australia article info Article history: Received 11 April 2014 Revised 13 June 2014 Accepted 23 June 2014 Available online 1 July 2014 Keywords: Gonadotropin releasing hormone Lipopeptide Vaccine T helper Murine immunogenicity abstract Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgi- cal castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH pep- tide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Com- plete Freund’s Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Our understanding and approaches to block or limit the release of reproductive hormones in an attempt to limit their influence on cancers has progressed to the development of immunotherapies. In spite of low immunogenicity, active immunisation against gonado- tropin releasing hormone (GnRH) has received considerable atten- tion because of potential applications in immunocontraception. An active area of research is the development of synthetic GnRH- based vaccines against reproductive hormone-dependent male and female cancers. 1 Studies have shown that immunisation against GnRH can reduce prostate levels of testosterone similar to surgical castration in numerous mammalian models, including mice and humans. 2,3 Immunisation of both males and females against GnRH can have a profound effect on fertility through the reduction in sex steroids and cessation of gametogenesis. Hence, there is a potential for GnRH therapeutics to be used either as a semi-permanent contraceptive or to extend the postnatal anovula- tory period. 4,5 In the 1970s a number of groups attempted to produce antibod- ies against GnRH by co-administration with Complete Freund’s Adjuvant (CFA). However, these attempts were unsuccessful in producing high anti-GnRH antibody titers. 6 Many small molecules like peptides (haptens) are successful immunogens only if they are attached to macromolecules (carriers). It is often necessary to modify these haptens for coupling with carriers to make a stable carrier–hapten complex. 7 Selection of a suitable carrier system, hapten density (hapten:carrier molar ratio), and conjugation meth- ods are critical for developing an optimal vaccine against hap- tens. 8,9 The first alum-adjuvanted vaccine which was successful in producing antibodies against GnRH was conjugated to a tetanus toxoid carrier. 10 A number of other fusion proteins have been pre- pared for the production of anti-GnRH antibodies that employ con- jugation to diphtheria toxoid (e.g. Improvac Ò ), keyhole limpet hemocyanin (e.g. GonaCon™) or ovalbumin, with many of them available commercially for veterinary use. 11–15 Another recombi- nant fusion protein consisting of GnRH and diphtheria toxoid was successful in phase I/II clinical trials. 16 Synthetic subunit vaccine systems eliminate the need for large carrier proteins, which are often associated with adverse effects in the host. 17 Since subunit vaccines are composed of different http://dx.doi.org/10.1016/j.bmc.2014.06.052 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +61 7 33469892; fax: +61 7 33654273. E-mail address: i.toth@uq.edu.au (I. Toth). Bioorganic & Medicinal Chemistry 22 (2014) 4848–4854 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc