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

阳离子肽基序是4至30个残基长的带正电荷的精氨酸（R）或赖氨酸（K）侧基簇，其大量存在于一些病毒和真核蛋白质中。阳离子肽将大的蛋白质或免疫刺激寡核苷酸递送到细胞中，因此代表了一种新型的、潜在的通用递送系统，用于将蛋白质和核苷酸以生物活性形式递送到胞质溶胶中。在拟议的项目中，我们将表征选定的阳离子结构域和DNA或RNA为基础的核苷酸之间的复合物。我们将测量合成的阳离子肽或具有阳离子基序的天然蛋白质与合成寡核苷酸、聚I/C或质粒DNA的定量相互作用。将表征肽/核苷酸复合物的生物物理参数（例如大小、稳定性、蛋白酶和核酸酶抗性）。将在四个系统中研究这些复合物递送至免疫系统的刺激：（i）将检测其对先天免疫系统的细胞（DC、NK细胞、NKT细胞）的佐剂作用作为诱导性细胞因子释放和表面标志物谱的调节。来自正常或突变（KO）小鼠的DC、NK细胞和NKT细胞将用于这些研究，这些小鼠缺乏其相关信号传导途径的先天性和/或适应性免疫系统的组分。(ii)将通过测定特异性、引发或加强（记忆）的CD 8 + T细胞群的数量、出现动力学和寿命，研究与核苷酸复合的阳离子/抗原融合肽引发特异性CD 8 + T细胞应答。(iii)将表征用于在不同抗原呈递细胞中的MHC I类限制性表位呈递的阳离子/抗原融合肽（作为与核苷酸的复合物递送）的加工。该分析将包括HBsAg衍生肽，其含有两种不同的Kb结合表位，这些表位仅由外源性或内源性HBsAg加工而成。(iv)阳离子蛋白与DNA疫苗的复合物形成将用于构建多价疫苗。将研究编码与代表抗原2的阳离子肽/蛋白质复合的抗原1的DNA疫苗的免疫原性，以有助于引发CD 8 + T细胞应答的新型候选疫苗的合理设计。

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.