Mechanisms of adenovirus neutralization

摘要 腺病毒载体血管内给药有望提高患者的生存率 扩散性转移性癌症，并通过永久性纠正改善许多遗传性疾病 造血干细胞区室。尽管潜在地有利，但血管内递送使得 易受免疫系统的先天和适应性臂的体液成分影响的治疗性广告。 我们和其他人以前的广泛分析表明，凝血FX与基于HAdv-5的载体的结合 促进高效的肝细胞转导，引发肝毒性。此外，相对 HAdv-5中和抗体（NAb）在人群中的高流行率，促使积极的 开发基于不与FX相互作用并且表现出低表达的替代Ad血清型的治疗载体 NAb患病率。然而，我们的初步研究表明，既有中和作用，也有非中和作用， 中和体液免疫可显著加剧宿主的炎性抗病毒应答。我们发现 大多数缺乏HAdv-5中和活性的人血清仍然能够触发补体结合 导致免疫细胞产生增强的炎症细胞因子。此外，我们发现， 用腺病毒HAdv-11免疫小鼠触发非中和抗体的产生， 在补体固定上对遗传学上远距离HAdv-5病毒是高效的。基于这些发现，我们 提出了一个新的概念，隐蔽调理非中和抗体，或CON抗体，结合到广告后， 全身递送，触发病毒上的补体固定，并靶向Ad-免疫复合物（Ad-IC）， 免疫吞噬细胞，导致全身炎症急剧增强。的分子机制 介导预先存在的非中和免疫的免疫刺激特性及其对安全性的影响 系统性广告投放几乎是未知的。因此，在本项目的具体目标1中，我们将分析如何 遗传学上远距离的HAdv血清型触发针对HAdv-5的CON-Ab的产生。在第二阶段，我们将 确定CON-Ab介导的补体结合病毒和补体的结构基础， 介导的病毒中和。在特定目标3中，我们将确定特定的细胞类型及其受体， 在体内隔离Ad-IC;在具体目标4中，我们将确定负责Ad-IC的特定信号通路。 加剧对Ad-IC的炎症反应，并开发靶向药理学方法来改善 在基因转移和癌症治疗模型中系统性Ad递送的安全性。拟议的研究将提供 对先天性和适应性免疫以及宿主抗病毒防御的基本功能的新的机械见解， 以及允许开发新的患者分层工具和药理学方法， 提高依赖于治疗性广告的全身递送的临床干预的安全性。

ABSTRACT Intravascular administration of adenovirus (Ad) vectors holds great promise for improving survival of patients with disseminated metastatic cancer and ameliorating numerous genetic diseases through permanent correction of the hematopoietic stem cell compartment. Although potentially advantageous, intravascular delivery makes therapeutic Ads vulnerable to humoral components of the innate and adaptive arms of the immune system. Extensive previous analyses by us and others showed that binding of coagulation FX to HAdv-5-based vectors in the blood facilitates highly efficient hepatocyte transduction, triggering hepatotoxicity. Furthermore, a relatively high prevalence of HAdv-5-neutralizing antibodies (NAbs) in the human population, prompted active development of therapeutic vectors based on alternate Ad serotypes that don't interact with FX and exhibit low NAb prevalence. Our preliminary studies indicate however, that both pre-existing neutralizing and non- neutralizing humoral immunity can significantly exacerbate the host inflammatory antiviral response. We found that the majority of human sera that lack HAdv-5-neutralizing activity are still able to trigger complement fixation on the virus, leading to potentiated inflammatory cytokine production by immune cells. Moreover, we found that immunization of mice with adenovirus HAdv-11 triggers generation of non-neutralizing antibodies, which are highly efficient at complement fixation on phylogenetically-distant HAdv-5 virus. Based on these findings, we propose a novel concept of cryptic opsonizing non-neutralizing antibodies, or CON-Abs, which bind to Ad after systemic delivery, trigger complement fixation on the virus, and target Ad-immune complexes (Ad-ICs) to immune phagocytic cells, leading to drastically potentiated systemic inflammation. The molecular mechanisms mediating the immune-stimulatory properties of pre-existing non-neutralizing immunity and its effect on the safety of systemic Ad delivery are virtually unknown. Therefore, in Specific Aim 1 of this project we will analyze how phylogenetically-distant HAdv serotypes trigger generation of CON-Abs to HAdv-5. In Specific Aim 2, we will determine the structural bases for CON-Ab-mediated complement fixation on the virus and complement- mediated virus neutralization. In Specific Aim 3 we will determine specific cell types and their receptors that sequester Ad-ICs in vivo; and in Specific Aim 4 we will determine specific signaling pathways responsible for the exacerbated inflammatory response to Ad-ICs and develop targeted pharmacological approaches to improve the safety of systemic Ad delivery in gene transfer and cancer therapy models. The proposed studies will provide new mechanistic insights into fundamental functions of innate and adaptive immunity and host anti-viral defense, as well as allow for the development of novel patient stratification tools and pharmacological approaches to improve the safety of clinical interventions that rely on systemic delivery of therapeutic Ads.