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。每年大约有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，每年仅在非洲就有200万至300万儿童死于疟疾。本研究的总体目标是优化腺病毒（Ad）载体，以获得强大的抗疟疾免疫能力作为有效的疟疾疫苗。我们早期的研究表明，表达约氏疟原虫环孢子子（CS）抗原（AdPyCS）的重组Ad可诱导CD8+ T细胞介导的抗疟疾保护性免疫。然而，AdPyCS不能诱导高水平的疟疾寄生虫抗体反应。由于已知Ad会引发一种强烈的预先存在的抗Ad免疫，主要针对其衣壳蛋白，我们假设将外源表位插入衣壳蛋白会诱导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蛋白的免疫显性B细胞表位插入到两个不同的衣壳蛋白中，即一个在纤维中，另一个在六邻体中，作为转基因表达了与GFP融合的整个PyCS蛋白。对每一种衣壳修饰的Ad（PyCS+GFP）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的Ad（PyCS+GFP）可诱导最高水平的保护性抗疟疾免疫，以及抗疟疾的免疫应答。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的疫苗能产生最强的抗疟疾保护作用的原因。我们将首先确认Ad(PyCS+GFP)-F/B的保护能力，然后确定不同Ad载体诱导的疟疾特异性体液和细胞反应水平，并详细描述其反应。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PyCS蛋白的产生，PyCS蛋白包括来自转基因的CD4+表位。因此，在Aim 2中，我们将通过在Ad(PyCS+GFP)-F/B的各种衣壳中插入疟疾特异性CD4+表位来构建一种新的Ad载体，并确定在Ad衣壳蛋白中插入cs特异性CD4+表位是否会引发强烈的cs特异性CD4+ T细胞（辅助细胞）反应，从而进一步增强抗cs抗体反应。Ad的衣壳修饰可能使病毒从抗衣壳抗体中逃脱，并且衣壳修饰的Ad能够在体内维持其感染性。因此，在最后一篇文章中，我们将确定衣壳修饰的Ad的组织/细胞趋向性和细胞内运输，并与未修饰的Ad进行比较。公共卫生相关性：疟疾仍然是一种破坏性疾病。该项目的目的是开发一种新的基于腺病毒的疟疾疫苗，这种疫苗不仅可以引起强烈的疟疾特异性细胞反应，而且还可以引起强烈的疟疾特异性体液反应。同时诱导高水平的两支免疫反应应导致成功地诱导针对疟疾的强效保护性免疫，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.