Unmasking the roles of viral glycoproteins in oral transmission of KSHV

项目总结 全球每年报告的卡波西肉瘤(KS)新病例超过44,000例，其中84%发生在 非洲。这种和其他卡波西肉瘤相关疱疹病毒(KSHV)诱导的恶性肿瘤在 有获得性或医源性免疫缺陷的人。虽然在其他人体内也能检测到KSHV 体液，在有和无性传播感染风险的人群中唾液中频繁检测到它(例如， 儿童)表明口腔是初级习得的部位。然而，KSHV口服的作用机制 体内传播，特别是病毒进入所需的关键病毒包膜糖蛋白(GPS)； 仍然悬而未决。已经确定了几种KSHV与宿主的相互作用，但所有先前的实验都是 在体外进行，由于之前缺乏合适的动物模型，尚未在体内得到验证。穿过 与威斯康星州国家灵长类研究中心合作，我们的实验室可以访问公共 绒猴(Callithrix Jacchus，CJ)，一种新开发的KSHV非人类灵长类动物模型，易患 KSHV口腔感染，并在免疫抑制下获得KS样皮肤病变。这样做的目的是 应用是阐明在活体内启动原发口腔感染所需的最低GPS，作为 选择关键GPS以开发有效的预防疫苗候选。此应用程序构建在DR的基础上。 Ogebo最近完成的KSHV进入机制和疫苗的NCI K01 CA184388-05研究 发展。最近，我们在体外证明了KSHV糖蛋白Gh/gl在病毒感染过程中是必不可少的。 上皮细胞、内皮细胞和成纤维细胞，但不是B细胞。值得注意的是，我们和其他人还表明， 抗KSHV糖蛋白gB、Gh/gl和gpK8.1的单抗和多克隆抗体可中和KSHV感染 体外培养的人类细胞种类繁多。在这一成功的基础上，我们产生了KSHV缺失突变体 被认为对病毒进入至关重要的四种糖蛋白(gb、Gh/gl、gpK8.1)和各种单抗 特定于这些GPS。在这个项目中，我们将使用人体体外样本和CJ KSHV模型来测试 假设Gb和Gh/gl是KSHV在体内口服传播的关键。我们提议的前提是 基于强有力的证据：1)KSHV可以感染CJ，导致KS样皮肤病变；2)抗KSHV抗体 KSHV糖蛋白gB和Gh/gl在体外和体外均能中和KSHV感染。此外， 体外细胞和CJ对KSHV感染的通透性使这些平台成为检测KSHV的理想平台 全科医生对感染的要求。成功完成拟议的研究将阐明最低的KSHV 在体外和体内模型中初次感染所需的GPS，推进了我们定义 人类感染KSHV的最初步骤和抗体在预防早期感染KSHV中的作用 变速箱。这最终将为预防KSHV的预防性疫苗的设计和开发提供信息 感染及其相关癌症。

PROJECT SUMMARY More than 44,000 new cases of Kaposi sarcoma (KS) are reported globally each year, 84% of which occur in Africa. This and other Kaposi sarcoma-associated herpesvirus (KSHV)-induced malignancies predominate in people with acquired or iatrogenic immunodeficiencies. Although KSHV can be detected in other human body fluids, its frequent detection in saliva in groups both with and without risk of sexually transmitted infections (e.g., children) suggests that the oral cavity is the site of primary acquisition. However, the mechanism of KSHV oral transmission in vivo, particularly the critical viral envelope glycoproteins (gps) required for viral entry, remains unresolved. Several KSHV–host interactions have been identified, but all prior experiments were performed in vitro and have not been validated in vivo due to prior lack of an appropriate animal model. Through collaboration with the Wisconsin National Primate Research Center, our laboratory has access to the common marmoset (Callithrix jacchus, CJ), a recently developed KSHV non-human primate model that is susceptible to KSHV oral infection, and under immunosuppression acquires KS-like skin lesions. The objective of this application is to elucidate the minimum gps required to initiate primary oral infection in vivo, as a prerequisite to selecting key gps for developing an effective prophylactic vaccine candidate. This application builds on Dr. Ogembo’s recently completed NCI K01 CA184388-05 research on KSHV entry mechanisms and vaccine development. Recently, we showed that in vitro, the KSHV glycoprotein gH/gL is essential for viral infection of epithelial, endothelial, and fibroblasts cells, but not B cells. Notably, we and others have also shown that both monoclonal and polyclonal Abs to KSHV glycoproteins gB, gH/gL, and gpK8.1, can neutralize KSHV infection of diverse permissive human cells in vitro. Building on this success, we generated KSHV deletion mutants lacking the four glycoproteins thought to be critical for viral entry (gB, gH/gL, gpK8.1) and various monoclonal antibodies specific to these gps. In this project, we will use human ex vivo samples and the CJ KSHV model to test the hypothesis that gB and gH/gL are critical for KSHV in vivo oral transmission. The premise of our proposal is built on strong evidence that 1) KSHV can infect CJ, which develop KS-like skin lesions, and 2) Abs against the KSHV glycoproteins gB and gH/gL can neutralize KSHV infection in vitro and ex vivo. Furthermore, the permissiveness to KSHV infection of human cells ex vivo and CJ makes these platforms ideal to test the KSHV gp requirements for infection. Successful completion of the proposed study will elucidate the minimum KSHV gps required for primary infection in ex vivo and in vivo models, advancing our long-term goal of defining the initial steps in KSHV infection of humans and the role of antibodies in protecting against the early steps of KSHV transmission. This will ultimately inform design and development of prophylactic vaccines that can prevent KSHV infection and its associated cancers.