Novel Linear Peptide Universal Malaria Vaccines

描述(由申请人提供)：该项目广泛的长期目标是评估含有疟原虫通用T细胞表位和免疫优势B细胞表位的合成线性多肽嵌合体(LPCS)作为有效疟疾疫苗成分的潜力。我们建议研究在两种间日疟原虫裂殖子蛋白中发现的一系列混杂T细胞表位。这些假定的通用T细胞表位是通过对可溶性DRB1*分子进行体外多肽结合试验来鉴定的。分别测试了代表这两种蛋白质的完整氨基酸序列的几组合成多肽。通过设计包含来自两个特征蛋白之一的单个T细胞表位和来自环子孢子(CS)蛋白的NANP重复序列的LPCS作为标准B细胞表位，证实了混杂T细胞表位免疫诱导的辅助活性。在整个构建体组被测试后，免疫后产生了非常高的抗肽和抗寄生虫抗体以及细胞抗肽Th1和Th2细胞因子谱。此外，在典型无应答的BALB/c小鼠中，对NANP序列的遗传限制被克服。重要的是，LPC免疫所产生的抗体也能够抑制活子孢子对人肝细胞的侵袭。我们现在的目标是进一步研究这种方法对亚单位疟疾疫苗设计的体内相关性。将设计包含其他混杂T细胞表位的新多肽结构，从我们鉴定的那些表位中挑选出来，并在具有不同遗传背景的小鼠身上测试免疫原性。我们假设，这些混杂的T细胞表位中的一些实际上是通用的T细胞表位，因为它们能够结合几个MHC II类分子。设计多聚体和多物种构建体的LPC方法的简单性有助于筛选几个变量，包括：多个T细胞表位的物理结合，特定T细胞表位对Th1或Th2表型免疫反应极化的内在活性，不同B细胞表位的包含，以及T辅助细胞活性所需的最小T细胞表位的定义。这些研究将为设计和测试选定的基于疟原虫的LPCS在恒河猴身上的有效性奠定基础，恒河猴是一种接近人类的动物模型。潜在的LPC免疫原将在肽结合分析中进行评估，以确定它们与Mamu-DR等位基因结合的能力，然后在实验中分别感染食蟹猴和Coatneyi的猴的免疫原性和召回反应中进行评估。这些方法将为评估LPC方法作为临床试验的前进途径提供强有力的数据，无论是单独使用还是与其他疟疾疫苗平台结合使用。

DESCRIPTION (provided by applicant): The broad long-term objective of this project is to evaluate the potential of synthetic linear peptide chimeras (LPCs) containing Plasmodium universal T cell epitopes and immunodominant B cell epitopes as effective malaria vaccine components. We propose to study a series of promiscuous T cell epitopes that we have identified in two Plasmodium vivax merozoite proteins. These putative universal T cell epitopes were identified by using in vitro peptide-binding assays to soluble DRB1* molecules. Sets of synthetic peptides, representing the entire amino acid sequences of both proteins, were individually tested. Helper activity induced by immunization with promiscuous T cell epitopes was confirmed by designing LPCs containing individual T cell epitopes from one of the two characterized proteins and the NANP repeat sequence from the P. falciparum circumsporozoite (CS) protein, as a standard B cell epitope for these evaluations. Very high antipeptide and antiparasite antibodies and cellular anti-peptide Th1 and Th2 cytokine profiles were elicited after immunization with the entire group of constructs tested. Further, genetic restriction to the NANP sequence was overcome in the typically non-responder BALB/c mouse. Importantly, the antibodies elicited by the LPC immunizations were also able to inhibit the invasion of viable sporozoites to human hepatocytes. We now aim to investigate further the in vivo relevance of this approach for the design of subunit malaria vaccines. New peptide constructs will be designed containing other promiscuous T cell epitopes, selected from those we have identified, and tested for immunogenicity in mice with diverse genetic backgrounds. We hypothesize that a number of these promiscuous T cell epitopes are in fact universal T cell epitopes for their ability to bind several MHC class II molecules. The simplicity of the LPC method for designing multimeric and multispecies constructs facilitates the screening of several variables including: the physical association of more that one T cell epitope, the intrinsic activity of the particular T cell epitopes on the polarization of the immune response towards a Th1 or Th2 phenotype, the inclusion of different B cell epitopes, and the definition of minimal T cell epitopes required for T helper cell activity. These studies will form the foundation for designing and testing the effectiveness of selected Plasmodium-based LPCs in rhesus monkeys, an animal model close to humans. Potential LPC immunogens will be evaluated in peptide-binding assays for their ability to bind Mamu-DR alleles, and then for immunogenicity and recall responses in monkeys experimentally infected with P. cynomolgi and P. coatneyi: simian malaria models for P. vivax and P. falciparum, respectively. These approaches will provide strong data to evaluate the LPC approach as a ways forward for clinical trials, whether used singly or in combination with other malaria vaccine platforms.