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后会产生针对FVIII(FVIII)的抑制性抗体。抑制物的开发与较低的生活质量和增加的经济负担有关，目前被认为是血友病A治疗中最重要的并发症。此外，第八因子抑制物可出现在非血友病患者中，即获得性血友病A，在这种情况下可能会出现危及生命和肢体的并发症。严重血友病A的患者没有循环中的FVIII蛋白，因此无法正常识别FVIII为“自我”。因此，治疗替代的FVIII蛋白被免疫系统视为“外来的”。鉴于FVIII是一种免疫外来蛋白，对FVIII产生抗体反应似乎并不令人惊讶。然而，在啮齿动物和人类中，静脉注射可溶性蛋白通常都不能诱导免疫，往往会导致耐受状态。因此，FVIII的强大免疫原性是意想不到的。这笔赠款的核心问题是，FVIII为什么以及通过什么机制诱导免疫反应。这项建议有三个具体目标。在目标1中，通过在小鼠血友病A免疫原性模型中比较FVIII的免疫原性和FVIII的非活性形式，将FVIII的免疫原性与FVIII的非活性形式进行比较来表征FVIII对免疫系统的作用，即在凝块形成的炎症部位和FVIII从其大载体蛋白von Willebrand因子中释放的位置。在目标2中，将构建一种新的FVIII分子(HOVA)，其特征在于将卵清蛋白的T细胞表位插入到B区。目前已有多种针对该T细胞表位的免疫试剂，包括四聚体试剂和TCR转基因小鼠，可用于研究静脉注射HOVA后CD4+T细胞应答的机制。有证据表明，在手术或大出血等“危险信号”发生时及早暴露于FVIII会增加抗体产生的风险。在目标3中，Toll样受体激活在FVIII免疫中的作用将通过比较静脉注射HOVA后的CD4+T细胞反应以及在存在和不存在Toll样受体激动剂的情况下的最终抗体产生来解决。这些实验结果将有助于更深入地了解FVIII的实质免疫原性，并可能为未来设计免疫原性降低的新型FVIII分子提供见解。这一研究计划以及本提案中规定的正式课程和有组织的指导应为候选人提供必要的培训，使她成为一名独立的调查员。

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.