Optimizing adenoviral vector to elicit a potent anti-malaria immunity

描述（由申请人提供）：疟疾仍然是世界上一种毁灭性的传染病。每年约有3亿至5亿人受到感染，发病率和死亡率相对较高。事实上，世界卫生组织估计，每年仅在非洲就有200万至300万儿童死于疟疾。本提案的总体目标是优化腺病毒（Ad）载体，以引发强大的抗疟疾免疫作为一种有效的疟疾疫苗。我们早期的研究表明，表达主要疟疾抗原约氏疟原虫环子孢子（CS）抗原的重组Ad，AdPyCS，可以诱导由CD 8 + T细胞介导的保护性抗疟疾免疫。然而，AdPyCS不能诱导针对疟原虫的高水平抗体应答。因为已知Ad引发主要针对其衣壳蛋白的强的预先存在的抗Ad免疫，所以我们假设将外源表位插入衣壳蛋白导致诱导强的表位特异性体液应答。为了检验这一假设，我们将PyCS蛋白的免疫显性B细胞表位插入表达与GFP融合的完整PyCS蛋白的Ad的两种不同衣壳蛋白中，即一种在纤维中，另一种在六邻体中，作为转基因。在每种修饰的或未修饰的Ad（PyCS+GFP）的多次免疫后，我们发现在纤维中表达B表位的Ad（PyCS+GFP），Ad（PyCS +GFP）-F/B，诱导最高水平的保护性抗疟疾免疫以及抗疟疾体液应答。在该提案的目标1中，我们计划寻找Ad（PyCS+GFP）-F/B比其他基于Ad的疫苗能够诱导最强的抗疟疾保护的原因。我们将首先确认Ad（PyCS+GFP）-F/B的保护能力，然后确定由不同Ad载体诱导的疟疾特异性体液和细胞应答的水平，并详细表征它们的应答。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captain修饰的Ad，从而减少了包含来自转基因的CD 4+表位的PyCS蛋白的产生。因此，在目的2中，我们将通过将疟疾特异性CD 4+表位插入Ad（PyCS+GFP）-F/B的各种衣壳中来构建新型Ad载体，并确定将CS特异性CD 4+表位插入Ad衣壳蛋白中是否可以引发强烈的CS特异性CD 4 + T细胞（辅助）应答，从而进一步增强稳健的抗CS抗体应答。这可能是由于衣壳蛋白的修饰使病毒逃避了抗衣壳蛋白抗体的攻击，而衣壳蛋白修饰的Ad在体内仍能保持其感染性。因此，在最后一个目的中，我们将确定captain修饰的Ad的组织/细胞向性和细胞内运输，并与未修饰的Ad进行比较。公共卫生相关性：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，该疫苗不仅可以引起强烈的疟疾特异性细胞应答，而且可以引起强烈的疟疾特异性体液应答。同时诱导高水平的免疫应答的双臂应导致成功诱导针对疟疾的有效保护性免疫，从而最终将基于Ad的疟疾疫苗作为有希望的疫苗。

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.