Priming specific, murine CD8+ T cell responses by complexes of cationic/ antigenic fusion peptides with nucleic acids

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Cationic peptide motifs are 4 to 30 residue long clusters of positively charged arginine (R) or lysine (K) side groups that are abundantly present in some viral and eukaryotic proteins. Cationic peptides deliver large proteins or immune-stimulating oligonucleotides into cells and thus represent a novel, potentially universal delivery system for proteins and nucleotide into the cytosol in bioactive form. In the proposed project we will characterize the complexes between selected cationic domains and DNA-or RNA-based nucleotides. We will measure the quantitative interaction of either synthetic, cationic peptides, or natural proteins with cationic motifs with synthetic oligonucleotides, poly I/C or plasmid DNAs. Biophysical parameters (e.g. size, stability, protease and nuclease resistance) of the peptide/nucleotide complexes will be characterized. The stimulus that these complexes deliver to the immune system will be studied in four systems: (i) its adjuvant effect on cells of the innate immune system (DC, NK cells, NKT cells) will be detected as inducible cytokine release and modulation of the surface marker profile. DC, NK cells, and NKT cells from normal or mutant (KO) mice that lack components of the innate and/or adaptive immune system of their associated signalling pathways will be used for these studies. (ii) Priming of specific CD8+ T cell responses by cationic/antigenic fusion peptides complexed to nucleotides will be studied by determining the numbers, kinetic of appearance and longevity of specific, primed or boosted (memory) CD8+ T cell populations. (iii) Processing of cationic/antigenic fusion peptides (delivered as complexes with nucleotides) for MHC class I-restricted epitope presentation in different antigen presenting cells will be characterized. This analysis will include HBsAg-derived peptides that contain two different Kb-binding epitopes that are processed exclusively from either exogenous or endogenous HBsAg. (iv) Complex formation of cationic proteins with DNA vaccines will be used to constuct polyvalent vaccines. The immunogenicity of a DNA vaccine encoding antigen 1 complexed to cationic peptide(s)/proteins representing antigen 2 will be studied in an effort to contribute to the rational design of novel vaccine candidates that prime CD8+ T cell responses.