Peptide-protein microarrays for the simultaneous detection of pathogen infections.

We describe novel peptide-protein microarrays, which were fabricated using semicarbazide glass slides that permitted the immobilization of glyoxylyl peptides by site-specific ligation and the immobilization of proteins by physisorption. The arrays permitted the simultaneous serodetection of antibodies directed against hepatitis C virus (HCV core p21 15-45 peptide, NS4 1925-1947 peptide, core, NS3, NS4, and mixture of core, NS3, NS4, and NS5 antigens), hepatitis B virus (HBc, HBe, and HBs), human immunodeficiency virus (Gp41 and Gp120 for HIV-I and Gp36 for HIV-II), Epstein-Barr virus (VCAp18 153-176 peptide), and syphilis (rTpN47 and rTpN17) antigens using an immunofluorescence assay. Peptide-protein microarrays displayed high signal-to-noise ratios, sensitivities, and specificities for the detection of antibodies as revealed by the analysis of a collection of human sera referenced against these five pathogens.

Simultaneous lipidation of a characterized peptide mixture by chemoselective ligation.

The modification of a peptide antigen by a fatty acid such as palmitic acid is now recognized as a mean to induce cellular responses. Mixtures of lipopeptides, obtained by combining individually synthesized compounds, were shown to be promising synthetic vaccine candidates. Usually, in lipopeptide synthesis, the fatty acyl moiety is introduced on the crude peptide chain using solid-phase methods. The separation of the target compound from impurities by RP-HPLC is often complicated by the amphiphilic properties of lipopeptides and results in low overall yields. To overcome the difficulties associated with lipopeptide synthesis and mixture preparation, we have developed a method where the fatty acyl moiety is site-specifically and collectively introduced in solution onto a mixture of individually prepurified peptides. The lipidation is based on the quasistoichiometric and high-yielding ligation of a glyoxylyl lipid with hydrazinoacetyl peptides. The hydrazone constructs were prepared in a salt-free medium and could be isolated by direct lyophilization of the reaction mixture. This process is compatible with cysteinyl peptides, and no aggregation nor degradation could be observed.

Grafting of synthetic mannose receptor-ligands onto onion vectors for human dendritic cells targeting.

A practical preparation of onion vesicles targeted to dendritic cells involves the grafting of mannose-mimetic clusters, bearing a hydrazino group, onto the surface of onion vesicles containing an aldehyde functionalized lipid.