Mechanisms of adenovirus neutralization

摘要 腺病毒(Ad)载体血管内给药在改善患者存活率方面有很大希望 患有播散性转移性癌症并通过永久矫正改善多种遗传性疾病 在造血干细胞室里。尽管有潜在的优势，但血管内分娩使 治疗广告易受免疫系统先天和适应性手臂的体液成分的影响。 我们和其他人之前的广泛分析表明，凝血因子X与基于HAdV-5的载体结合 在血液中促进高效的肝细胞转导，引发肝脏毒性。此外，一个相对的 HAdV-5中和抗体(NAB)在人类人群中的高流行率，促使活动 基于不与FX相互作用的交替Ad血清型的治疗载体的开发 NAB流行率。然而，我们的初步研究表明，先前存在的中和和非 中和体液免疫可显著加剧宿主的炎症性抗病毒反应。我们发现 大多数缺乏HAdV-5中和活性的人血清仍然能够引发补体结合 导致免疫细胞产生炎性细胞因子。此外，我们发现， 用腺病毒HAdV-11免疫小鼠会引发非中和抗体的产生，这些抗体是 对系统发育上遥远的HAdV-5病毒进行高效的补体结合。基于这些发现，我们 提出了一种新的隐蔽调理非中和抗体的概念，即CON-Abs，它与Ad. 全身性传递，触发病毒上的补体结合，并靶向Ad-免疫复合体(Ad-IC)以 免疫吞噬细胞，导致严重的全身炎症。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 系统性的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 系统发育上相距遥远的HAdV血清型触发了针对HAdV-5的ConAb的产生。在具体目标2中，我们将 确定刀豆蛋白抗体介导的补体与病毒结合的结构基础 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；在特定的目标4中，我们将确定负责 加剧对Ad-ICs的炎症反应，并开发有针对性的药理学方法来改善 基因转移和肿瘤治疗模型中系统递送Ad的安全性。拟议的研究将提供 对先天免疫和获得性免疫的基本功能以及宿主抗病毒防御的新机械见解， 以及允许开发新的患者分层工具和药理学方法来 提高依赖系统投放治疗性广告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.