MODULAR CHIMERIC VACCINES TAILORED FOR MALARIA PARASITES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们已经报告了旨在确定多聚体多肽作为红细胞前疟疾疫苗递送系统的潜力的原理研究的证据。第一代多肽嵌合体，我们称之为线性多肽嵌合体(LPC)，被设计成包括代表具有良好特征的红细胞前B细胞表位的线性序列。对几种红细胞期候选疫苗的结构分析表明，保护性抗体主要识别呈现复杂三级结构的功能结构域。 为了证实我们提高疟疾候选疫苗免疫原性的策略也可以用于非线性结构域，我们设计了约氏疟原虫嵌合重组蛋白，该蛋白包含串联组装的自体混杂T细胞表位，并连接到特征良好的裂殖子表面蛋白1的羧基末端结构域，称为PyMSP119。合成的基因经过密码子优化后在大肠杆菌和重组产物中进行了表达，我们称之为重组模块化嵌合体(RMC)，用于免疫原性和 仅含有PyMSP119蛋白片段的对照蛋白具有保护作用。 包含自体T细胞表位并没有改变重组产物的完整性。相应地，T细胞表位被成功表达并能够诱导表位特异性T细胞反应。RMC在与同源P.yoelii株进行实验攻击后，对高度寄生虫血症和严重贫血都产生了强大的保护作用。最重要的是，我们发现RMC诱导的功能性抗体比对照蛋白诱导的抗体更有效地抵御异源攻击。根据实验证据，约氏疟原虫红细胞前体型 (LPC)和红细胞期(RMC)重组嵌合蛋白诱导保护性免疫，我们决定评估LPC和RMC在单一免疫原中的潜在协同效应。我们构建了一个编码氨基末端红细胞前LPC和羧基末端红细胞RMC的杂合合成基因。初步数据表明，该杂交疫苗能够诱导小鼠产生无菌免疫。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have reported proof of principle studies designed to determine the potential of multimeric polypeptides as a delivery system for pre-erythrocytic malaria vaccines. The first generation of polypeptide chimeras, which we have called linear peptide chimeras (LPC), was designed to include linear sequences representing well-characterized pre-erythrocytic B cell epitopes. Structural analyses of several erythrocytic stage vaccine candidates revealed that protective antibodies predominantly recognize functional domains that exhibit complex tertiary structure. To confirm that our strategy to improve immunogenicity of malaria vaccine candidates can also be used for non-linear structured domains, we designed P. yoelii chimeric recombinant protein comprising autologous promiscuous T cell epitopes assembled in tandem and linked to carboxyl terminal domain of well-characterized merozoite surface protein 1 known as PyMSP119. The synthetic gene was codon-optimized for expression in E. coli and the recombinant product, that we have called Recombinant Modular Chimera (RMC), used for comparative experiments of immunogenicity and protective efficacy with control protein that only included PyMSP119 protein fragment. The inclusion of autologous T cell epitopes did not alter the integrity of the recombinant product. Relevantly, T cell epitopes were successfully expressed and able to induce epitope-specific T cell responses. RMC generated robust protection to both hyper-parasitemia and severe anemia after experimental challenge with homologous P. yoelii strain. Most importantly, we found that RMC induced functional antibodies that protected against heterologous challenge more efficiently than antibodies induced by control protein. Based on experimental evidence that P. yoelii pre-erythrocytic (LPC) and erythrocytic stage (RMC) recombinant chimeric proteins induced protective immunity, we decided to evaluate the potential synergistic effect of combining LPC and RMC in a single immunogen. We created a hybrid synthetic gene encoding an amino-terminal pre-erythrocytic LPC and a carboxyl terminal erythrocytic RMC. Preliminary data indicate that such hybrid vaccine is able to induce sterilizing immunity in mice.