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

摘要：爱泼斯坦-巴尔病毒(Epstein-Barr Virus，EBV)是一个主要的全球健康问题，因为它与 每年全世界青少年传染性单核细胞增多症和20万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，但目前还没有获得许可的预防性EBV 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，还没有疫苗 候选人已经诱导出中和抗体(NAB)来完全阻断体内的EBV感染。实现以下目标的方法 EBV疫苗的开发在一定程度上受到病毒基因组的致癌潜力和缺乏 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战略来开发一个安全的 和有效的候选疫苗，将多达5个EBV糖蛋白整合到单个Epstein-Barr病毒中- 类颗粒(EBV-LP)。EBV糖蛋白gp350、gb、gp42和Gh/gl复合体，它们是 EBV的附着、融合和进入宿主细胞是激发体液/抗体的有吸引力的目标- 中介反应。我们的团队和其他人已经证明，针对这些糖蛋白的抗体可以中和病毒 体外和体内感染。因此，它们的纳入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抗原的组合还没有在Pre- 临床或临床试验。我们将生成包含多达五种糖蛋白组合的EBV-LP 改良的牛痘安卡拉病毒。我们将确定EBV-LP产生NAB应答的有效性 野生型小鼠，然后测试这些抗体中和人上皮细胞90%EBV感染的能力 并在人源化小鼠模型中预防体内人类B细胞的感染(目标1)。 由于EBV是亲人的，我们将使用EBV同源的恒河猴淋巴病毒(RhLCV)作为 替代病毒研究恒河猴(RM)体内疫苗效力，被认为是最相关的 总结人类EB病毒感染关键特征的动物模型。重要的是，RM口服接种 重组人LCV表现出与EBV+人类相似的急性症状和病毒脱落，以及发生 免疫抑制下持续感染后的淋巴瘤。我们将评估我们的EBV-LP的能力- 基于或类似的基于重组人LCV-LP的疫苗，以诱导可预防或限制感染的NAB，无/低EBV 血液、脾细胞或淋巴结中的DNA，以及免疫活性和无EBV+癌 与特征良好的基于EBV gp350的疫苗相比，免疫受损的RM疫苗(目标2)。我们的中央 假设EBV-LPs/rhLCV-LPs将产生保护性的抗EBV/rhLCV糖蛋白NAB应答 目的：预防EBV/rhLCV在体内外的感染。我们希望我们的方法能够提供一条通往 预防性EB病毒疫苗的研究新药应用。我们还将首次定义 在体外和体内诱导无菌NAB所需的最低EBV/rhLCV糖蛋白。这将推进我们的 开发预防EBV感染和EBV相关疾病和癌症的疫苗的长期目标。

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.