Immune Evasion Mechanisms of KSHV Complement and Fc-Receptor Proteins

项目摘要 卡波西肉瘤相关疱疹病毒（KSHV）是卡波西肉瘤的致病因子， 如皮肤或粘膜表面的肿瘤，以及两种类型的B细胞淋巴瘤，多中心Castleman病 和原发性渗出性淋巴瘤由于没有疫苗或治疗方法可以成功预防或 根除KSHV，部分原因是由于其逃避宿主免疫防御的能力，它仍然是一个重要的公众 健康负担。虽然B细胞淋巴瘤是罕见的，但超过44，000例新的卡波西肉瘤病例， 每年在全球范围内报道。因此，迫切需要阐明KSHV逃逸的机制 人体免疫系统疱疹病毒，包括KSHV，与它们的宿主共同进化了数百万年， 劫持宿主调节因素来伪装自己并逃避广泛的宿主防御措施。 值得注意的是，像其他疱疹病毒一样，KSHV已经获得了一种补体控制蛋白（KCP），发现其调节 宿主补体介导的免疫防御。不幸的是，KCP在生物学相关环境中的研究 由于缺乏生产KCP无效KSHV和测试KCP效果的系统， 病毒感染细胞上的补体相互作用。为了解决这些缺陷，我们产生了重组KSHV， 使用新建立的KSHV细菌人工染色体（BAC）， 技术，以及新的抗KCP抗体来表征突变病毒。在辅导期间（K99） 阶段，我将使用这些资源来描述KCP在逃避补体控制和感染中的作用。 除KCP外，KSHV还编码一种糖蛋白K1，其具有获得性免疫球蛋白结构域， 片段可结晶受体（FcR）功能。其他疱疹病毒中的类似结构域已被证明可以 作为通常结合免疫细胞上存在的FcR的宿主抗病毒抗体的干扰受体;然而， K1在KSHV的Fc介导的免疫逃避中的作用还没有研究。因此，在 K99阶段，我建议产生和表征抗K1抗体，并表征我们最近 产生稳定的rKSHVΔK1生产细胞系。这些资源将在独立（R 00）阶段使用 以表征K1的Fc型相互作用和结合能力。我也会研究 K1干扰宿主FcR激活使用一种新的报告分析。最后，我将评估抗K1的疗效 抗体阻断Fc介导的免疫逃避。因此，本文提出的研究将提供一个更好的 了解KCP和K1在宿主免疫逃避中的作用。这项工作将大大促进发展 探讨预防和控制KSHV感染及致病的有效策略。此外，研究和 为K99阶段提出的培训将提供病毒基因组学/蛋白质组学方面的经验和知识， 免疫生物学和病毒肿瘤学，以及大量可验证的数据和初步数据， 许多未来的研究，这将提供一个坚实的基础上，建立我的独立研究生涯。

PROJECT ABSTRACT Kaposi sarcoma-associated herpesvirus (KSHV) is the causal agent of Kaposi sarcoma, a cancer that appears as tumors on the skin or mucosal surfaces, and two types of B-cell lymphomas, multicentric Castleman disease and primary effusion lymphoma. As there are no vaccines or therapeutic treatments to successfully prevent or eradicate KSHV, in part due to its ability to evade its host’s immune defenses, it remains a significant public health burden. Although B-cell lymphomas are rare, more than 44,000 new cases of Kaposi sarcoma are reported globally each year. Thus, there is a critical need to elucidate the mechanisms by which KSHV escapes the human immune system. Herpesviruses, including KSHV, co-evolved with their hosts over millions of years, hijacking host regulatory factors to camouflage themselves and evade a wide range of host defense measures. Notably, like other herpesviruses, KSHV has acquired a complement control protein (KCP) found to modulate the host complement-mediated immune defense. Unfortunately, studies of KCP in a biologically relevant setting have been hindered by the absence of a system to produce KCP-null KSHV and test the effects of KCP– complement interactions on virus-infected cells. To address these deficiencies, we generated recombinant KSHV (rKSHV) lacking KCP (rKSHVΔKCP) using newly established KSHV bacterial artificial chromosome (BAC) technology, as well as novel anti-KCP antibodies to characterize the mutant virus. During the mentored (K99) phase, I will use these resources to characterize the role of KCP in evading complement control and infection. In addition to KCP, KSHV encodes a glycoprotein, K1, that has acquired immunoglobulin structure domains with fragment crystallizable receptor (FcR) functions. Similar domains in other herpesviruses have been shown to act as distractor receptors for host antiviral antibodies that typically bind FcRs present on immune cells; however, the role of K1 in Fc-mediated immune evasion by KSHV has not been examined. Therefore, toward the end of the K99 phase, I propose to generate and characterize anti-K1 antibodies and characterize our recently generated stable rKSHVΔK1 producer cell lines. These resources will be utilized in the independent (R00) phase to characterize the Fc-type interactions and binding capabilities of K1. I will also study the mechanism by which K1 interferes with host FcR activation using a novel reporter assay. Finally, I will evaluate the efficacy of anti-K1 antibodies in blocking Fc-mediated immune evasion. Thus, the studies proposed herein will provide a better understanding of the role of KCP and K1 in host immune evasion. This work will greatly enhance the development of effective strategies to prevent and control KSHV infection and pathogenesis. Furthermore, the research and training proposed for the K99 phase will provide experience and knowledge in viral genomics/proteomics, immunobiology, and viral oncology, as well as a significant body of publishable data and preliminary data for many future studies, which will provide a solid foundation upon which to build my independent research career.