A multivalent prophylactic and therapeutic vaccine against EBV infection and EBV-associated malignancies

摘要：EB病毒（EBV）是一个主要的全球性健康问题，因为它与以下疾病有关： 每年全球青少年传染性单核细胞增多症和> 200，000例儿童和成人癌症病例。 尽管与EBV感染相关的发病率和死亡率很高， 疫苗因此，迫切需要一种安全有效的EBV预防性疫苗。到目前为止，没有疫苗 候选人已经引发中和抗体（nAb）以在体内完全阻断EBV感染。方法来 EBV疫苗的开发部分受到病毒基因组的致癌潜力和缺乏免疫原性的限制。 动物模型来测试候选疫苗。这个拟议的项目将使用一种新的策略来开发一种安全的 和有效的候选疫苗，将多达五种EB病毒糖蛋白整合到单个EB病毒中- EB病毒样颗粒（EBV-LP）。EBV糖蛋白gp 350、gB、gp 42和gH/gL复合物，它们是EBV感染所必需的。 EBV附着、融合和进入宿主细胞是激发体液/抗体的有吸引力的靶点， 介导的反应。我们的研究小组和其他人已经证明，这些糖蛋白的抗体可以中和病毒 体外和体内感染。因此，将它们包括在内对于开发有效的预防性疫苗至关重要， 防止病毒感染。据我们所知，这种EBV抗原的组合尚未在临床试验前进行过测试。 临床或临床试验。我们将使用以下方法产生含有多达五种糖蛋白的组合的EBV-LP： 改良的安卡拉牛痘病毒。我们将确定EBV-LPs在小鼠中产生nAb应答的功效。 野生型小鼠，然后测试这些抗体中和>90%的人上皮细胞EBV感染的能力。 和B细胞，并在人源化小鼠模型中体内预防人B细胞的感染（目的1）。 由于EBV是嗜人的，我们将使用EBV同源的恒河猴淋巴隐病毒（rhLCV）作为免疫原性载体。 用于研究恒河猴（RM）体内疫苗功效的替代病毒，被认为是最相关的 动物模型重现了人类EBV感染的关键特征。重要的是，RM经口接种 rhLCV表现出与EBV+人类相似的急性症状和病毒脱落，以及 淋巴瘤后持续感染免疫抑制。我们将评估我们的EBV-LP的能力， 基于rhLCV-LP的或类似的基于rhLCV-LP的疫苗，以引发可以预防或限制感染的nAb， 血液、脾细胞或淋巴结中的DNA，以及免疫活性和 免疫受损的RM，与充分表征的基于EBV gp 350的疫苗相比（目的2）。我们的中央 假设EBV-LP/rhLCV-LP将产生保护性抗EBV/rhLCV糖蛋白nAb应答 在体外和体内预防EBV/rhLCV感染。我们希望我们的方法能够提供一条通往 用于预防性EBV疫苗的研究性新药申请。我们还将首次定义 在体外和体内引发灭菌nAb所需的最小EBV/rhLCV糖蛋白。这将推动我们的 长期目标是开发预防EBV感染和EBV相关疾病和癌症的疫苗。

ABSTRACT: Epstein-Barr virus (EBV) represents a major global health problem, as it is associated with infectious mononucleosis in adolescents and >200,000 pediatric and adult cancer cases worldwide each year. Despite the high morbidity and mortality associated with EBV infection, there is no licensed prophylactic EBV vaccine. Thus, a safe and effective EBV prophylactic vaccine is urgently needed. To date, no vaccine candidate has elicited neutralizing antibodies (nAbs) to completely block EBV infection in vivo. Approaches to EBV vaccine development have been limited in part by the oncogenic potential of the viral genome and a lack of animal models to test vaccine candidates. This proposed project will use a novel strategy to develop a safe and effective vaccine candidate that incorporates up to five EBV glycoproteins into a single Epstein-Barr virus- like particle (EBV-LP). The EBV glycoproteins gp350, gB, gp42, and gH/gL complex, which are essential for EBV attachment, fusion, and entry into host cells, are attractive targets for provoking a humoral/antibody- mediated response. Our group and others have shown that antibodies to these glycoproteins neutralize viral infection in vitro and in vivo. Thus, their inclusion is critical for developing an effective prophylactic vaccine that protects against viral infection. To our knowledge, this combination of EBV antigens has not been tested in pre- clinical or clinical trials. We will generate EBV-LPs containing combinations of up to five glycoproteins using modified vaccinia Ankara virus. We will determine the efficacy of the EBV-LPs to generate nAb responses in wild-type mice, then test the ability of these antibodies to neutralize >90% of EBV infection of human epithelial and B cells in vitro and to prevent infection of human B cells in vivo in a humanized mouse model (Aim 1). Because EBV is human-tropic, we will use the EBV-homologous rhesus lymphocryptovirus (rhLCV) as a surrogate virus to study vaccine efficacy in vivo in rhesus macaques (RM), considered the most relevant animal model that recapitulates key features of human EBV infection. Importantly, RM orally inoculated with rhLCV exhibit acute symptoms and viral shedding similar to EBV+ humans, as well as development of lymphoma after persistent infection under immunosuppression. We will evaluate the ability of our EBV-LP- based or analogous rhLCV-LP-based vaccines to elicit nAbs that can prevent or limit infection, with no/low EBV DNA in blood, splenocytes, or lymph nodes, and absence of EBV+ cancer in immunocompetent and immunocompromised RM, compared to well-characterized EBV gp350-based vaccines (Aim 2). Our central hypothesis is that EBV-LPs/rhLCV-LPs will generate protective anti-EBV/rhLCV glycoprotein nAb responses to prevent EBV/rhLCV infection in vitro and in vivo. We expect our approach to provide a path to an investigational new drug application for a prophylactic EBV vaccine. We will also define, for the first time, the minimum EBV/rhLCV glycoproteins required to elicit sterilizing nAbs in vitro and in vivo. This will advance our long-term goal of developing vaccines that prevent EBV infection and EBV-associated diseases and cancers.