Lipopeptides containing 2-(palmitoylamino)-6,7-bis(palmitoyloxy)heptanoic acid: synthesis, stereospecific stimulation of B-lymphocytes and macrophages and adjuvanticity in vivo and in vitro

Abstract

Lipopeptides, carrying the N-terminal lipoamino acid 2-(palmitoylamino)-6,7-bis(palmitoyloxy) heptanoic acid (Pam3Adh-OH, 1), were obtained by solid-phase synthesis and by synthesis in solution. 2-Amino-6,7-dihydroxyheptanoic acid (Adh) can be regarded as a methylene analogue of S-glycerylcysteine, the N-terminal amino acid of lipoprotein from the outer cell membrane of Escherichia coli (a methylene group substitutes for the sulfur atom). The lipopeptides Pam3Adh-Ser-Ser-Asn-Ala 2a-d, in which the four possible stereoisomers of Pam3Adh-OH (2S,6S)-1 (1a), (2S,6R)-1 (1b), (2R,6S)-1 (1c), and (2R,6R)-1 (1d) are linked to the naturally occurring sequence Ser-Ser-Asn-Ala of the N-terminus of lipoprotein, and also Pam3Adh-Ser-(Lys)4 [2S,6S)-3), with a peptide part rendering the molecule water soluble, were capable of stimulating murine splenocytes polyclonally in vitro, as determined in a proliferation assay and in a hemolytic plaque assay against trinitrophenylated sheep erythrocytes. The diastereomers (2S,6S)-2 and (2R,6S)-2 with S-configurated C-6 were more active than the diastereomers (2S,6R)-2 and (2R,6R)-2 with R-configurated C-6; a change of the configuration at C-2 had less effect on the stimulatory activity. (2S,6S)-2 and (2S,6S)-3 are potent immunoadjuvants. A significantly enhanced primary immune response against trinitrophenylated sheep erythrocytes was obtained in vitro at lipopeptide concentrations of about 5 micrograms/mL and an immunization dose of 10(7) sheep erythrocytes/mL. Balb/c mice, which were immunized with a mixture of ovalbumin and (2S,6S)-2 or (2S,6S)-3, respectively, had a substantially higher antiovalbumin titer 28 days after immunization than mice which had received ovalbumin, (2S,6S)-2 or (2S,6S)-3 alone. Finally, the novel lipopeptides constitute potent macrophage activators: (2S,6S)-3 was able to induce tumor cytotoxicity against the tumor cell line L929 in bone marrow derived macrophages.

Palmitoyl Derivatives of GpMBP Epitopes: T-Cell Response and Peptidases Susceptibility

“…Lipopeptide 3, bearing a N -palmitoyllysine residue, was prepared using Fmoc- The two additional palmitoyl residues were introduced, still in solid phase, on the hydroxyl functions of the S-(2,3-dihydroxypropyl) present on the side chain of Cys, by recirculation of palmitoyl chloride, as previously described. 18,20 As control peptide, we synthesized Met-Ser-GpMBP(74-85) (4), elongating the wild-type sequence 1 with Met and Ser at the N-terminus.…”

Self-Adjuvanting Lipopeptide Vaccines

“…Pam H H S SKKKK [58,61] *R or R/S isomers (For P3Adh-SK4 the S isomer was superior [56]. This isomer has the same configuration as the P3C-SK4 R isomer).…”

Lipid-based Self-Adjuvanting Vaccines

“…In a head to head comparison of different adjuvants mixed with a malarial epitope for CTL induction following a single subcutaneous immunisation, Pam3Cys-Ser-(Lys)4 either as an admix or when covalently coupled to the epitope, was found to be superior to other experimental formulations [83]. Much effort has been directed towards optimisation of the peptide component of Pam3Cys-containing adjuvants [98][99][100][101] and examination of other chemical derivatives [102] for maximum biological function.…”