Optimizing adenoviral vector to elicit a potent anti-malaria immunity

描述(申请人提供)：疟疾仍然是世界上一种毁灭性的传染病。每年约有3-5亿人感染，发病率和死亡率相对较高。事实上，世界卫生组织估计，仅在非洲，每年就有200-300万儿童死于疟疾。这项建议的总体目标是优化腺病毒(Ad)载体，以诱导强大的抗疟疾免疫作为有效的疟疾疫苗。我们早期的研究表明，表达疟疾主要抗原--约氏疟原虫环子孢子(CS)抗原的重组Ad AdPyCS可以诱导由CD8+T细胞介导的保护性抗疟疾免疫。然而，AdPyCS不能诱导针对疟疾寄生虫的高水平抗体反应。由于已知的是，Ad能够诱导一种强大的、主要针对其衣壳蛋白的、预先存在的抗Ad免疫，因此我们假设，在衣壳蛋白中插入一个外源表位可以诱导出一种强大的表位特异性体液反应。为了验证这一假设，我们将PyCS蛋白的免疫优势B细胞表位插入到Ad的两种不同的衣壳蛋白中，即一种在纤维中，另一种在六角体中，表达融合到GFP的整个PyCS蛋白，作为转基因。在对每个衣壳修饰或未修饰的Ad(PyCS+GFP)进行多次免疫后，我们发现在纤维中表达B表位的Ad(PyCS+GFP)-F/B诱导了最高水平的保护性抗疟疾免疫以及抗疟疾体液反应。在本提案的目的1中，我们计划寻找为什么Ad(PyCS+GFP)-F/B比其他基于Ad的疫苗具有更强的抗疟疾保护作用的原因。我们将首先确定Ad(PyCS+GFP)-F/B的保护能力，然后测定不同Ad载体诱导的疟疾特异性体液和细胞反应水平，并详细描述它们的反应。我们的下一个假设是，重复免疫或预先存在的免疫仍然影响衣壳修饰的Ad，减少包括转基因CD4+表位在内的PYCS蛋白的产生。因此，在目标2中，我们将通过将疟疾特异性的CD4+表位插入到Ad(PyCS+GFP)-F/B的不同衣壳中来构建新型的Ad载体，并确定将CS特异性的CD4+表位插入到Ad衣壳蛋白中是否可以诱导强烈的CS特异性的CD4+T细胞(辅助)应答，从而进一步增强强大的抗CS抗体应答。可能是衣壳修饰的Ad使病毒逃脱了抗衣壳抗体，并且衣壳修饰的Ad能够在体内保持其感染性。因此，在最后一个目标中，我们将确定衣壳修饰的Ad的组织/细胞趋向性和细胞内转运，并与未修饰的Ad进行比较。与公共卫生相关：疟疾仍然是一种毁灭性的疾病。该项目的目的是开发一种新的基于腺病毒的疟疾疫苗，这种疫苗不仅能引起强烈的疟疾特异性细胞反应，而且能引起强大的疟疾特异性体液反应。同时诱导两支高水平的免疫反应应能成功诱导出针对疟疾的有效保护性免疫，从而最终使基于Ad的疟疾疫苗成为一种有希望的疫苗。

DESCRIPTION (provided by applicant): Malaria is still a devastating infectious disease of the world. Approximately 300 - 500 million people become infected yearly, with relatively high rates of morbidity and mortality. In fact, the WHO estimates that 2 - 3 million children die of malaria in Africa alone, every year. The overall aim of this proposal is to optimize adenoviral (Ad) vector in order to elicit a robust anti-malarial immunity as an effective malaria vaccine. Our earlier studies have shown that a recombinant Ad expressing a major malaria antigen, circumsporozoite (CS) antigen of Plasmodium yoelii, AdPyCS, could induce a protective anti-malarial immunity, which is mediated by CD8+ T cells. However, AdPyCS was unable to induce high levels of antibody response against malaria parasites. Because Ad is known to elicit a strong pre-existing anti-Ad immunity that is primarily against its capsid proteins, we hypothesized that the insertion of a foreign epitope into the capsid proteins results in an induction of a robust epitope-specific humoral response. To test this hypothesis, we have inserted an immunodominant B cell epitope of the PyCS protein into two different capsid proteins, i.e. one in the fiber and another in the hexon, of Ad that expresses an entire PyCS protein fused to GFP, as a transgene. After multiple immunization of each capsid-modified, or unmodified Ad(PyCS+GFP), we found that Ad(PyCS+GFP) expressing the B epitope in the fiber, Ad(PyCS+GFP)-F/B, induces a highest level of protective anti-malarial immunity, as well as anti-malarial humoral response. In Aim 1 of this proposal, we plan to seek the reasons why Ad(PyCS+GFP)-F/B can induce a strongest anti-malarial protection than other Ad-based vaccines. We will first confirm the protective capacity of Ad(PyCS+GFP)-F/B and then determine the levels of malaria-specific humoral and cellular responses induced by the different Ad vectors and characterize their responses in detail. Our next hypothesis is that a repeated immunization or a pre-existing immunity still affects capsid-modified Ad, reducing the production of the PyCS protein that includes a CD4+ epitope from the transgene. In Aim 2, therefore, we will construct a novel Ad vector by inserting a malaria-specific CD4+ epitope into various capsids of Ad(PyCS+GFP)-F/B, and determine whether the insertion of a CS-specific CD4+ epitope into the Ad capsid protein could elicit a strong CS-specific CD4+ T cell (helper) response, thereby enhancing further a robust anti-CS antibody response. It is possible that capsid modification of Ad makes the virus escape from anti-capsid antibodies, and that capsid-modified Ad is able to maintain its infectivity in vivo. In the last Aim, therefore, we will determine the tissue/cellular tropism and the intracellular trafficking of capsid-modified Ad and compare to those of unmodified Ad. PUBLIC HEALTH RELEVANCE: Malaria is still a devastating disease. The aim of this project is to develop a new adenovirus- based malaria vaccine that can elicit not only a strong malaria-specific cellular response, but also a robust malaria-specific humoral response. The simultaneous induction of high levels of both arms of the immune responses should lead to a successful induction of a potent protective immunity against malaria, thus ultimately placing Ad-based malaria vaccine, as a promising vaccine.