Geometry of a TLR2-Agonist-Based Adjuvant Can Affect the Resulting Antigen-Specific Immune Response.

Targeted delivery of otherwise nonimmunogenic antigens to Toll-like receptors (TLRs) expressed on dendritic cells (DCs) has proven to be an effective means of improving immunogenicity. For this purpose, we have used a branched cationic lipopeptide, R4Pam2Cys, which is an agonist for TLR2 and enables electrostatic association with antigen for this purpose. Here, we compare the immunological properties of ovalbumin formulated with different geometrical configurations of R4Pam2Cys. Our results demonstrate that notwithstanding the presence of the same adjuvant, branched forms of R4Pam2Cys are more effective at inducing immune responses than are linear geometries. CD8+ T-cell-mediated responses are particularly improved, resulting in significantly higher levels of antigen-specific cytokine secretion and cytolysis of antigen-bearing target cells in vivo. The results correlate with the ability of branched R4Pam2Cys conformations to encourage higher levels of DC maturation and facilitate superior antigen uptake, leading to increased production of proinflammatory cytokines. These differences are not attributable to particle size because both branched and linear lipopeptides associate with antigen-forming complexes of similar size, but rather the ability of branched lipopeptides to induce more efficient TLR2-mediated cell signaling. Branched lipopeptides are also more resistant to trypsin-mediated proteolysis, suggesting greater stability than their linear counterparts. The branched lipopeptide facilitates presentation of antigen more efficiently to CD8+ T cells, resulting in rapid cell division and upregulation of early cell surface activation markers. These results as well as cognate recognition of Pam2Cys by TLR2 indicate that the adjuvant's efficiency is also dependent on its geometry.

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.

Inclusion of TAT and NLS sequences in lipopeptide molecules generates homogenous nanoparticles for gene delivery applications.

PURPOSES: The objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity. METHODS: Two lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene. RESULTS: Our findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties. Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6-8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0. CONCLUSION: These results indicate that the lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.