Differential reactivity of maleimide and bromoacetyl functions with thiols: application to the preparation of liposomal diepitope constructs.

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine, and cysteine residues present at the N-terminus of peptides) was investigated in aqueous media. These studies were performed (i) with water-soluble functionalized model molecules, i.e., polyoxyethylene-based spacer arms that could also be coupled to lipophilic anchors destined to be incorporated into liposomes, and (ii) with small unilamellar liposomes carrying at their surface these thiol-reactive functions. Our results indicate that an important kinetic discrimination (2-3 orders of magnitude in terms of rate constants) can be achieved between the maleimide and bromoacetyl functions when the reactions with thiols are performed at pH 6.5. The bromoacetyl function which reacts at higher pH values (e.g., pH 9.0) retained a high chemoselectivity; i.e., under conditions where it reacted appreciably with the thiols of, e.g., HS-peptides, it did react with other nucleophilic functions such as alpha- and epsilon-amino groups or imidazole, which could also be present in peptides. This differential reactivity was applied to design chemically defined and highly immunogenic liposomal diepitope constructs as synthetic vaccines, i.e., vesicles carrying at their surface two different peptides conjugated each to a specific amphiphilic anchor. This was realized by coupling sequentially at pH 6.5 and 9.0 two HS-peptides to preformed vesicles containing lipophilic anchors functionalized with maleimide and bromoacetyl groups [Boeckler, C., et al. (1999) Eur. J. Immunol. 29, 2297-2308].

Design of highly immunogenic liposomal constructs combining structurally independent B cell and T helper cell peptide epitopes.

We have designed liposomal diepitope constructs that allow the physical combination, within the same vesicle, of B and Th epitopes as structurally separate entities. The immune response against such constructs was explored using TPEDPTDPTDPQDPSS (TPE), a B cell epitope originating from a Streptococcus mutans surface adhesin and QYIKANSKFIGITEL (QYI), a "universal" Th epitope from tetanus toxin. The two peptides were linked to the outer surface of small (diameter approximately 100 nm) unilamellar liposomes by covalent conjugation to two different anchors. To that end we have developed a strategy that allows the controlled chemical coupling of TPE and QYI, functionalized at their N terminus with a thiol, to preformed liposomes containing thiol-reactive derivatives of phosphatidylethanolamine and the lipopeptide S-[2,3-bis (palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-cysteinyl-alanyl-gly cine (Pam3CAG), respectively. This synthetic construct (administered i.p. to BALB/c mice) induced highly intense (titers > 20,000), anamnestic and long-lasting (over 2 years) immune responses, indicating that this strategy is successful. Two parameters were of prime importance to elicit this response with our liposomal diepitope constructs: (1) the simultaneous expression of B and Th epitopes on the same vesicle, and (2) the lipopeptide Pam3CAG anchor of the Th epitope QYI could not be replaced by a phosphatidylethanolamine anchor (a lesser immune response was observed). Analysis of the antibody response revealed a complex pattern; thus, besides the humoral response (production of IgG1, IgG2a, IgG2b) a superposition of a T-independent (TI-2 type) response was also found (IgM and IgG3). These results indicate that liposomal diepitope constructs could be attractive in the development of synthetic peptide-based vaccines.