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

The lipopeptide, R(4)Pam(2)Cys, 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 R(4)Pam(2)Cys 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 > R(4)Pam(2)Cys = alum. Each of the three adjuvants facilitated IgG class-switching with significantly more IgG1 elicited by OVA when formulated with R(4)Pam(2)Cys. The effects of substituting naturally occurring l-stereoisomers of the cationic residues within R(4)Pam(2)Cys with d-stereoisomers revealed that substitution did not affect the ability of R(4)Pam(2)Cys 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.