title: Synthesis of thiol-reactive lipopeptide adjuvants. Incorporation into liposomes and study of their mitogenic effect on mouse splenocytes.
abstract: Synthetic analogues of triacylated and diacylated lipopeptides derived from the N-terminal domain of respectively bacterial and mycoplasmal lipoproteins are highly potent immunoadjuvants when administered either in combination with protein antigens or covalently linked to small peptide epitopes. Because of their amphipathic properties, lipopeptides, such as S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-glycine (Pam(3)CAG), can be conveniently incorporated into liposomes and serve as anchors for antigens that are linked to them. To design vaccination constructs based on synthetic peptides and liposomes as vectors. we have accordingly synthesized a series of lipopeptides that differ by the number (Pam(3)C vs Pam(2)C) and nature of the acyl chains (palmitoyl vs oleoyl) and by the presence at their C-terminus of thiol-reactive functions, such as maleimide or bromoacetyl. When incorporated into liposomes, these latter functionalized lipopeptides allow, in aqueous media, a well controlled chemoselective conjugation of HS-peptides to the surface of the vesicles. Using a BALB/c mice splenocyte proliferation assay ([(3)H]thymidine incorporation), we have measured the lymphocyte activation potency of the different lipopeptides. We found that, compared to their free (emulsified) forms, the liposomal lipopeptides were endowed with enhanced mitogenic activities; i.e., up to 2 orders of magnitude for Pam(3)CAG which was more potent than Pam(2)CAG. The impact of functionalization on the cellular activity of Pam(3)CAG was dependent on the thiol-reactive group introduced: whereas the bromoacetyl derivative retained its full activity, the presence of a maleimide group virtually abolished the lymphocyte activation of the lipopeptide. Finally, the substitution of saturated palmitoyl chains by unsaturated oleoyl chains was inhibitory. Thus, thiol-reactive Ol(3)CAG derivatives were the least active mitogens in our assay. Taken together, our findings are of importance for the further optimization of antigen-specific liposomal-based synthetic vaccines; the bromoacetyl derivative of Pam(3)CAG should be a promising lipopeptide derivative serving as an anchor for peptide epitopes while retaining its lymphocyte activation activity.
authors.affiliation: Laboratoire de Chimie Bioorganique, Faculté de Pharmacie, UMR 7514 CNRS-Université Louis Pasteur, 74 route du Rhin, 67400 Strasbourg-Illkirch, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Roth
authors.forename: Socorro
authors.initials: S
authors.lastname: Espuelas
authors.forename: Christine
authors.initials: C
authors.lastname: Thumann
authors.forename: Benoît
authors.initials: B
authors.lastname: Frisch
authors.forename: Francis
authors.initials: F
authors.lastname: Schuber
mesh_terms: D000276:Adjuvants, Immunologic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D008074:Lipoproteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D015394:Molecular Structure; D013154:Spleen / Q000166:cytology / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D000276:Adjuvants, Immunologic; D008074:Lipoproteins; D008081:Liposomes; D008934:Mitogens; D013438:Sulfhydryl Compounds