Lipopeptide vaccines

The lipopeptide vaccine described in this study consists of a CD4+ helper T-cell epitope ([T]) and a B-cell epitope ([B]). These two epitopes are separated by a lysine residue (K) to which is attached the lipid moiety via two serine residues (Fig. IB). The CD4+ T-helper epitope KLIPNASLIENCTKAEL used is derived from the fusion protein of the morbillivirus canine distemper virus (34) and is recognized by T cells from BALB/c and C57BL6 mouse strains (4). The B-cell epitope is LHRH and has the sequence HWYSGLRPG. The presence of anti-LHRH antibodies can render vaccinated animals sterile. 

Fig. 1. Structural representations and fidelity of synthesis of lipopeptide and nonlipidated peptides. (A) Structure of the lipopeptide vaccines palmitic acid, Pam. (B) HPLC chromatograms of purified lipopeptides. Analysis by HPLC was performed on a Waters HPLC System using a Vydac C4 column with 0.1% TFA in water and 0.1% TFA in acetonitrile as a gradient mobile phase. A flow rate of 1 mL/min was used at a gradient of 2%/min. Chromatograms were obtained by the detection of absorbance at a wavelength of 214 nm. (C) Mass spectral analysis of purified lipopeptides. Lipopeptides were analyzed by mass spectrometry using an Agilent 1100 Series LCM/MSD ion trap.

Dissolve peptide and lipopeptide vaccines in saline to a concentration of 200 nmol/mL. If necessary, encourage dissolution by warming in a water bath and/or by sonication. Do not mix by pipet if the inoculant is not soluble this can result in insoluble peptide or lipopeptide being trapped in the pipet tip. 

Fig. 11.2 Mono- (Ghielmetti et al. 2005), di- (Buwitt-Beckmann et al. 2005), and tri-acylated (Bessler et al. 1985) examples of lipopeptide vaccines...

Gahery-Segard H, Pialoux G, Charmeteau B, Sermet S, Poncelet H, Raux M, Tartar A, Levy JP, Gras-Masse H, Guillet J-G (2000) Multiepitopic B- and T-cell responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine. J Virol 74(4) 1694-1703... 

WiesmuUer K-H, Bessler WG, Jung G (1992) Solid phase peptide synthesis of lipopeptide vaccines eliciting epitope-specific B-, T-helper and T-killer cell response. Int J Pept Protein Res 40 255-260. 

Bettahi I, Dasgupta G, Renaudet O et al (2009) Antitumor activity of a self-adjuvanting glycol-lipopeptide vaccine bearing B cell, CD4-I- and CD8-I- T cell epitopes. Cancer Immunol Immunother 58 187-200... 

Renaudet O, Dasgupta G, Bettahi I et al (2010) Linear and branched glyco-lipopeptide vaccines follow distinct cross-presentation pathways and generate different magnitudes of antitumor immunity. PLoS One 5 ell216... 

Deres, K., Schild, H., Wiesmuller, K. H., Jung, G., Rammensee, H. G., 1989, In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 342 56 W. 

The two examples from our work we are going to describe below are the design and study of liposomal diepitope constructs combining either (i) B and T-helper (Th) peptide epitopes, which induced particularly powerful humoral responses (21) (Fig. 3) or (ii) CTL and Th epitopes, which provided a powerful antitumor vaccine (74) (Fig. 4). For the production of these constructs we have conjugated peptides that contain a cysteine residue either at the N- or C-terminus, to the surface of preformed liposomes by reaction with thiol reactive functionalized phospholipids and/or PamaCys lipopeptide anchors (Fig. 2). To that end, we have developed strategies that give, in aqueous media, high... 

Figure 4 Design of a chemically defined diepitope liposomal anticancer vaccine. Small unilamellar liposomes (PC/PG/Chol 75/20/50 molar ratio diameter 65nm) containing 5mol% of the synthetic thiol-reactive lipopeptide adjuvant anchor Pam3CSS-Mal were reacted, at 25°C and pH 6.5, with equimolar quantities of the peptides ErbB2 (p63-71), derivatized with a CG linker at its N-terminus, and HA307-319, derivatized with a C-linker at its C-terminus. Abbreviations PC, phosphatidylcholine PE, phosphatidylethanolamine SUV, small unilamellar vesicles. Source From Refs. 11, 74.

Tosi PF, Radu D, Nicolau C. Immune response against the murine MDRI protein induced by vaccination with synthetic lipopeptides in liposomes. Biochem Biophys Res Commun 1995 212 494. 

Babu IS, et al. Priming for virus-specific CD8+ but not CD4+ C54otoxic T lymphocytes with synthetic lipopeptide is influenced by acylation units and liposome encapsulation. Vaccine 1995 13 1669. 

Conlan JW, et al. Immunization of mice with lipopeptide antigens encapsulated in novel liposomes prepared from the polar lipids of various Archaeobacteria elicits rapid and prolonged specific protective immunity against infection with the facultative intracellular pathogen. Listeria monocytogenes. Vaccine 2001 19 3509. 

Synthesis of Toll-Like Receptor-2 Targeting Lipopeptides as Self-Adjuvanting Vaccines... 

There are many techniques that can be employed to assess the immuno-genicity of a vaccine candidate, and laboratories interested in pursuing this objective should also explore other assays suited to their purposes. In this model, vaccine immunogenicity is measured by determining levels of anti-LHRH antibodies induced in mice immunized with lipopeptide or a nonlipi-dated peptide control. It is important to correlate vaccine immunogenicity with biological function, and we measure the reproductively capability of vaccinated female mice as an indication of vaccine efficacy. Techniques that determine testosterone and oestrogen levels are also useful. 

Le Gal, F.A., Prevost-Blondel, A., Lengagne, R., et al. (2002) Lipopeptide-based melanoma cancer vaccine induced a strong MART-27-35-cytotoxic T lymphocyte response in a preclinal study. Int. J. Cancer 98(2), 221-227. 

Chua, B.Y., Healy, A., Cameron, P.U., et al. (2003) Maturation of dendritic cells with lipopeptides that represent vaccine candidates for hepatitis C virus. Immunol. Cell Biol. 81(1), 67-72. 

Zeng, W., Ghosh, S., Lau, Y.F., Brown, L.E., and Jackson, D.C. (2002) Highly immunogenic and totally synthetic lipopeptides as self-adjuvanting immunocontra-ceptive vaccines. J. Immunol. 169(9), 4905 1912. 

Interactions between lipids and receptors for structurally conserved pathogen-associated molecular patterns, most commonly the Toll-like receptors (TLRs), have been studied in detail for their involvement in adjuvant activity. TLRs appear to play an important role in the functioning of many adjuvants and the most important TLRs for lipopeptide or glycolipopeptide vaccines are TLR1, TLR2, TLR4, and TLR6 (van Duin et al. 2006). 

BenMohamed L, Thomas A, Bossus M, Brahimi K, Wubben J, Gras-Masse H, Druilhe P (2000) High immunogenicity in chimpanzees of peptides and lipopeptides derived from four new Plasmodium falciparum pre-erythrocytic molecules. Vaccine 18(25) 2843-2855... 

Brayden DJ, Baird AW (2001) Microparticle vaccine approaches to stimulate mucosal immunisation. Microb Infect 3(10) 867-876 Brewer JM (2006) (How) do aluminium adjuvants work Immunol Lett 102(1) 10-15 Buwitt-Beckmann U, Heine H, Wiesmuller KH, Jung G, Brock R, Akira S, Ulmer AJ (2005) Toll-like receptor 6-independent signaling by diacylated lipopeptides. Eur J Immunol 35(1) 282-289... 

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