Unmasking the roles of viral glycoproteins in oral transmission of KSHV

项目概要本申请是根据特别关注通知（NOSI）提交的。 标识为“NOT-CA-22-057”。全球每年报告的卡波西肉瘤（KS）新发病例超过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 恶性肿瘤在具有获得性或医源性免疫缺陷的人中占优势。虽然KSHV可以 在其他人体体液中检测到，在有和没有风险的人群中，其在唾液中的频繁检测 性传播感染（例如，儿童）表明口腔是主要的获得部位。 然而，KSHV在体内经口传播的机制，特别是关键的病毒包膜， 病毒进入所需的糖蛋白（GPS）仍然没有解决。几种KSHV-宿主相互作用 已经确定，但所有先前的实验都是在体外进行的，并且由于先前的实验， 缺乏合适的动物模型。通过与威斯康星州国家灵长类动物研究中心的合作 中心，我们的实验室已经获得了共同的绒猴（Callithrix jacchus，CJ），最近开发的KSHV 对KSHV口腔感染易感并处于免疫抑制下的非人灵长类动物模型获得 KS样皮肤病变此应用程序的目的是阐明启动主要所需的最低GPS 体内口腔感染，作为选择用于开发有效预防性疫苗的关键GPS的先决条件 候选人此应用程序基于奥根博博士最近完成的NCI K 01 CA 184388 -05研究， KSHV进入机制和疫苗开发。最近，我们发现在体外，KSHV糖蛋白 gH/gL是病毒感染上皮细胞、内皮细胞和成纤维细胞所必需的，但不是B细胞所必需的。值得注意的是， 其他人也表明，抗KSHV糖蛋白gB、gH/gL和 gpK8.1能在体外中和KSHV对多种可接受的人细胞的感染。在这一成功的基础上，我们 产生的KSHV缺失突变体缺乏被认为对病毒进入至关重要的四种糖蛋白（gB，gH/gL， gpK8.1）和对这些gps特异的各种单克隆抗体。在这个项目中，我们将使用人类离体 样本和CJ KSHV模型，以检验gB和gH/gL对KSHV体内口服至关重要的假设 传输我们的建议的前提是建立在强有力的证据，1）KSHV可以感染CJ， KS样皮肤病变，和2）针对KSHV糖蛋白gB和gH/gL的Ab可以中和KSHV感染， 体外和离体。此外，对离体和CJ的人细胞的KSHV感染的容许性使得这些细胞在体外被KSHV感染。 这是检测KSHV gp感染要求的理想平台。成功完成拟议的研究将 阐明了体外和体内模型中原发性感染所需的最低KSHV gps，推进了我们的研究。 长期目标是确定KSHV感染人类的初始步骤以及抗体在保护人类中的作用。 与KSHV传播的早期阶段相对抗这将最终为预防性药物的设计和开发提供信息。 可以预防KSHV感染及其相关癌症的疫苗。

PROJECT SUMMARY This application is being submitted in response to the Notice of Special Interest (NOSI) identified as “NOT-CA-22-057.” 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.