Structure-function relationships of protein-lipopeptide complexes and influence on immunogenicity

The lipopeptide, R₄Pam₂Cys, associates electrostatically with soluble protein antigens and significantly enhances their ability to induce protective humoral and cell-mediated responses. We demonstrate that antibody titers elicited by the antigen ovalbumin (OVA) associated with R₄Pam₂Cys are higher than those elicited by OVA in the presence of alum and comparable to those elicited by OVA formulated with complete Freund's adjuvant (CFA). The hierarchy of anti-OVA antibody avidities was CFA > R₄Pam₂Cys = alum. Each of the three adjuvants facilitated IgG class-switching with significantly more IgG1 elicited by OVA when formulated with R₄Pam₂Cys. The effects of substituting naturally occurring L-stereoisomers of the cationic residues within R₄Pam₂Cys with D-stereoisomers revealed that substitution did not affect the ability of R₄Pam₂Cys to stimulate dendritic cell maturation or its ability to elicit antibody production when used as an adjuvant. Minor detrimental effects were, however, observed in the ensuing CD8⁺ T cell responses suggesting that the use of D-amino acids affects antigen processing and presentation pathways involved in generation of cell-mediated immunity at least when facilitated through TLR2. Both D- and L-forms were found to be resistant to digestion by trypsin, indicating resistance of the branched structure to protease activity.