Mechanisms of Immunogenicity of Factor VIII

描述（由申请人提供）：大约30%的严重血友病A患者在输注fVIII后产生因子VIII （fVIII）的抑制性抗体。抑制剂的发展与生活质量降低和经济负担增加有关，目前被认为是血友病a治疗中最重要的并发症。此外，因子VIII抑制剂可发生在非血友病患者，即获得性血友病a，其中可能存在危及生命和肢体的并发症。严重的A型血友病患者没有循环中的fVIII蛋白，因此不会形成对fVIII正常的“自我”识别。因此，治疗性替代fVIII蛋白被免疫系统视为“外来”。鉴于fVIII是一种免疫外源蛋白，对fVIII产生抗体反应似乎并不奇怪。然而，在啮齿类动物和人类中，静脉注射可溶性蛋白通常不能诱导免疫，往往会引起耐受性状态。由于这个原因，流感病毒的大量免疫原性是出乎意料的。这项拨款的核心问题是fVIII为什么以及通过什么机制诱导免疫反应。本提案有3个具体目标。在Aim 1中，在小鼠血友病a免疫原性模型中，通过比较fVIII的免疫原性和无活性形式的fVIII的免疫原性来表征fVIII向免疫系统呈递的生理部位，即血栓形成的炎症部位和fVIII从其大载体蛋白血管性血友病因子中释放出来的部位。在Aim 2中，将构建一个新的fVIII分子（HOVA），该分子具有插入B结构域的卵白蛋白的良好特征的T细胞表位。目前已有大量针对该T细胞表位的免疫试剂，包括四聚体试剂和TCR转基因小鼠，可用于研究CD4+ T细胞对静脉输注HOVA的反应机制。有证据表明，在手术或大出血等“危险信号”出现时，早期暴露于fVIII会增加抗体产生的风险。在Aim 3中，将通过比较CD4+ T细胞对静脉输注HOVA的反应，以及在存在和不存在toll样受体激动剂的情况下最终产生的抗体，来解决toll样受体激活对fVIII免疫的作用。这些实验的结果将允许更深入地了解fVIII的实质性免疫原性，并可能为未来设计免疫原性降低的新型fVIII分子提供见解。该研究计划以及该提案中列出的正式课程和结构化指导应该为候选人提供必要的培训，使其成为一名独立的研究者。

DESCRIPTION (provided by applicant): Inhibitory antibodies to factor VIII (fVIII) develop in approximately 30% of patients with severe hemophilia A in response to infusions of fVIII. Inhibitor development is associated with a lower quality of life and an increased economic burden and is currently is considered the most significant complication of the management of hemophilia A. Additionally, factor VIII inhibitors can occur in nonhemophiliacs, i.e. acquired hemophilia A, in which there can be life and limb- threatening complications. Patients with severe hemophilia A have no circulating fVIII protein and thus normal recognition of fVIII as "self" does not develop. Accordingly, the therapeutic replacement fVIII protein is seen as "foreign" by the immune system. Given that fVIII is an immunologically foreign protein, it may not seem surprising that an antibody response to fVIII would occur. However, in both rodents and humans, intravenous injection of soluble proteins usually fails to induce immunity and often induces a tolerogenic state. For this reason, the substantial immunogenicity of fVIII is unexpected. The central question of this grant is why, and by what mechanism, does fVIII induce an immune response. There are 3 Specific Aims in this proposal. In Aim 1, the role of the physiological site of fVIII presentation to the immune system, i.e. a site of inflammation where a clot is forming and where fVIII is released from its large carrier protein von Willebrand factor, will be characterized by comparing the immunogenicity of fVIII with an inactive form of fVIII in a murine hemophilia A immunogenicity model. In Aim 2, a novel fVIII molecule (HOVA) will be constructed that has a well characterized T cell epitope of ovalbumin inserted into the B domain. A wide array of immunologic reagents specific for this T cell epitope, including tetramer reagents and TCR transgenic mice, are available to investigate the mechanism of CD4+ T cell response to intravenous infusion of HOVA. There is evidence that early exposure to fVIII at the time of a "danger signal" such as surgery or a major bleed increases the risk of antibody development. In Aim 3 the role of toll like receptor activation on fVIII immunity will be addressed by comparing the CD4+ T cell response to intravenous infusion of HOVA as well as the ultimate antibody production in the presence and absence of toll like receptor agonists. The results of these experiments will allow for a deeper understanding of the substantial immunogenicity of fVIII and might provide insights for future design of novel fVIII molecules with decreased immunogenicity. This research plan along with the formal coursework and structured mentorship laid out in this proposal should provide the candidate with the training necessary to establish her as an independent investigator. PUBLIC HEALTH RELEVANCE: Patients with hemophilia A have a deficiency in the blood coagulation protein factor VIII. Some patients with hemophilia A develop antibodies to factor VIII that worsens their bleeding problems and makes therapy difficult and expensive. In this project, we will develop a better understanding of the immune response to the factor VIII protein, which may lead to better treatment alternatives.