RESEARCH ARTICLE Design, synthesis and characterisation of mannosylated ovalbumin lipid core peptide self-adjuvanting vaccine delivery system Pavla Simerska & Zyta Maria Ziora & Vincent Fagan & Daryn Goodwin & Farrah Edrous & Istvan Toth Published online: 18 September 2013 # Controlled Release Society 2013 Abstract Peptide-based vaccine delivery can be hampered by rapid peptidase activity and poor inherent immunogenicity. The self-adjuvanting lipid core peptide system (LCP) has been shown to confer improved stability and immunogenicity on peptide epitopes of group A Streptococcus , Chlamydia , hook- worm, and malaria pathogens. However, various diseases, including cancer, still require targeted delivery of their vaccine candidates. For this reason, we have selected two model peptides (ovalbumin CD4 + and/or CD8 + T cell epitopes), and incorporated two or four copies of either epitope into our LCP vaccine. Optimised glycosylation of ovalbumin pep- tides yielded 46 % when microwave-assisted double coupling with 2 eq of carbohydrate derivative, activated by N ,N - diisopropylethylamine and (O -benzotriazol-1-yl)-N ,N ,N ′,N ′- tetramethyluronium hexafluorophosphate, was performed. All ovalbumin peptides were successfully synthesised and puri- fied in 11–55 % yields by Fmoc- or Boc-chemistry using solid-phase peptide synthesis. The mannosylated ovalbumin peptides were nontoxic to human erythrocytes in haemolytic assay (<2 % haemolysis) and showed increased (up to 20-fold) stability in plasma. Keywords Vaccine development . Peptide synthesis . Ovalbumin . Glycosylation . In vitro assay Introduction A large number of potent and highly specific therapeutic peptides and proteins have been found that have the potential to cure or prevent a variety of diseases [1–3]. Synthetic subunit peptide vaccines are popular due to their safety, ease of large-scale production, purity and lack of side effects when compared to whole-organism vaccines. However, peptide an- tigens are generally unstable in vivo and have limited immu- nogenicity because they do not stimulate an innate immune response. The immunostimulatory properties of peptide vac- cines can be improved by co-administration with an adjuvant, and/or incorporation into a self-adjuvanting delivery system. Many strong adjuvants are used in animal models, but are too toxic for use in humans. Aluminium-based adjuvants are one of the few adjuvants licensed for human application, though they are quite weak and unstable [4]. Recently, other adju- vants, such as MF59, ASO4 and virosomes, have also been used in vaccine delivery [5]. The lipid core peptide (LCP) system was designed to be a self-adjuvanting delivery system, avoiding the need for a con- ventional adjuvant. The LCP system is composed of lipoamino acids (α-amino acids with long alkyl side chains) and a poly- lysine, or carbohydrate core, providing branching for attach- ment of multiple copies of peptide antigens [6]. LCP vaccines can be readily synthesised by solid-phase peptide synthesis (SPPS), a common technique for peptide production. The efficacy of the LCP system has previously been demonstrated in animal models against Chlamydia [7], group A Streptococ- cus [8], hookworm [9], and malaria [10]. While many advan- tages have been described about the lipidation of peptides, increased lipophilicity leads to poor water solubility, an impor- tant factor in vaccine delivery [6]. Glycosylation of peptides has been reported to improve their stability and uptake, as well as enhance water solubility, transport across biological barriers [11] and allow targeting to specific sites [12–15]. P. Simerska (*) : Z. M. Ziora : V. Fagan : D. Goodwin : F. Edrous : I. Toth School of Chemistry and Molecular Biosciences, The University of Queensland, Cooper Road, St Lucia, QLD 4072, Australia e-mail: p.simerska@uq.edu.au I. Toth Pharmacy Australia Centre of Excellence, School of Pharmacy, The University of Queensland, Cornwall Street, Woolloongabba, QLD 4102, Australia Drug Deliv. and Transl. Res. (2014) 4:246–255 DOI 10.1007/s13346-013-0173-8