Please cite this article in press as: Zeng, W., et al., Lipidation of intact proteins produces highly immunogenic vaccine candidates. Mol. Immunol. (2010), doi:10.1016/j.molimm.2010.10.003 ARTICLE IN PRESS G Model MIMM-3569; No. of Pages 7 Molecular Immunology xxx (2010) xxx–xxx Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Lipidation of intact proteins produces highly immunogenic vaccine candidates Weiguang Zeng a,∗ , Emily M. Eriksson a , Andrew Lew b , David C. Jackson a,∗∗ a Department of Microbiology & Immunology, The University of Melbourne, Parkville 3010, Victoria, Australia b Walter and Eliza Hall Institute, Parkville 3010, Victoria, Australia article info Article history: Received 29 July 2010 Received in revised form 7 October 2010 Accepted 10 October 2010 Available online xxx Key words: Protein Lipidated protein Vaccine Pam2Cys Antibody Covalent coupling abstract In this study we investigate the feasibility of generating self-adjuvanting vaccines capable of inducing high titre antibody responses following the covalent attachment of the TLR2 agonist Pam 2 Cys to intact proteins. Three Pam 2 Cys-based lipid moieties were prepared which contain a solubilising spacer com- posed of either lysine residues or polyethyleneglycol. A model protein, hen egg white lysozyme (HEL), was lipidated individually with each of these lipid modules and the immunogenicity of the lipidated species studied in mice by measuring antibody responses. We found that lipidated HEL elicited antibodies which is much stronger than the responses obtained when the HEL was administered in Freund’s adjuvant or in Alum. Little or no antibody was elicited by the lipidated HEL in CD4 T cell-deficient mice indicating that the antibody response is T cell dependent. Furthermore, the lipidated protein elicited similar antibody responses in two different strains of mice indicating that sufficient helper T cell epitopes are available to enable antibody production across the histocompatability barrier. In a similar way, lipidated bovine insulin was found to be highly immunogenic in mice despite the largely conserved sequences of bovine and murine insulin. The results provide evidence that lipidation of proteins provides a simple and safe method for the manufacture of soluble self-adjuvanting protein-based vaccines. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved. 1. Introduction The advances made in immunology and recombinant pro- tein manufacturing technologies have allowed a more rational approach to the design of vaccines with a result that protein and subunit vaccines are now competing with empirically designed, traditional, vaccines. Vaccines against hepatitis B and human papil- loma viruses (HPV) are protein-based and available in the market with more protein-based vaccine candidates such as the envelope glycoproteins of HIV (Zhou et al., 2007), pertactin from Bordetella pertussis (Leef et al., 2000), and hD52, a target protein for ovarian cancer (Byrne et al., 2005) being evaluated in clinical trials with more appearing almost on a daily basis. Although protein antigens can induce antibody responses that in many cases are suitable for protection from disease, they usually have to be inoculated in the presence of an adjuvant in order to produce the antibody titres that are necessary for efficacy. Adju- vant research is also a vigorous area of endeavor because many are toxic causing potentially dangerous site reactions and as a con- sequence few are currently licensed for use in humans. For these ∗ Corresponding author. Tel.: +61 3 83449942. ∗∗ Corresponding author. Tel.: +61 3 83449940. E-mail addresses: weiguang@unimelb.edu.au (W. Zeng), davidcj@unimelb.edu.au (D.C. Jackson). reasons huge efforts are being made to discover new, effective and safe adjuvants and significant progress in the rational design of adjuvants is being made notably in the case of Toll-like receptor agonists. This laboratory, among others, has investigated the util- ity of incorporating dipalmitoyl-S-glyceryl cysteine (Pam 2 Cys) into peptides and demonstrating their potential as vaccines against viral (Deliyannis et al., 2006; Alphs et al., 2008; Day et al., 2007; Lau et al., 2006), and bacterial (Batzloff et al., 2006; Jackson et al., 2004) infec- tions, model tumours (Baz et al., 2008; Jackson et al., 2004) as well as abrogating normal physiological functions such as hormone- mediated reproduction (Zeng et al., 2005, 2002, 2007). The rational for using Pam 2 Cys is that it is an agonist for TLR-2 and targets cells, including dendritic cells (DCs) that express this receptor on their surface. Because TLR-2 is also an endocytic receptor (Takeda and Akira, 2007), Pam 2 Cys facilitates transport of Pam 2 Cys-containing molecules into the DC and then causes cell activation through sig- nal transduction via MyD88 (Zeng et al., 2010; Takeda and Akira, 2007). The clear potential of Pam 2 Cys in allowing the manufacture of prophylactic and therapeutic peptide-based vaccines prompted us to extend the utility to subunit or protein-based vaccines. In this study we investigated the feasibility of generating a self-adjuvanting protein-based vaccine that is capable of inducing antibody responses by the covalent attachment of the TLR2 agonist Pam 2 Cys to an intact protein. Protein-based vaccine manufacture is greatly facilitated by the availability of soluble protein antigen and an ability to carry out manufacturing steps in aqueous media. 0161-5890/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2010.10.003