Rapid development and testing of Zika virus vaccine candidates

项目摘要 寨卡病毒（ZIKV）是一种由伊蚊传播的包膜黄病毒， 无症状或轻度登革热样疾病。2015年初，巴西爆发了一场大规模的ZIKV疫情， 传播到美洲超过25个国家。令人担忧的是，ZIKV巴西疫情已经 与受感染母亲所生婴儿的数千例小头畸形病例有关，导致世卫组织 最近宣布国际关注的突发公共卫生事件。因此，控制工作是 迫切需要控制疫情，避免进一步蔓延到其他国家。没有ZIKV 已经开发了疫苗，尽管用于相关登革病毒（DENV）的疫苗是基于 膜（M）和包膜（E）蛋白诱导保护性免疫，在临床上处于高级阶段。 其中一种（Dengvaxia）刚刚在墨西哥、巴西和菲律宾获得批准。痘苗病毒 （VACV）用于根除天花，天花是由天花病毒（一种相关的痘病毒）引起的疾病。VACV具有 也被成功地用作复制能力或缺陷型病毒载体，用于开发 有效的人类和动物疫苗，因为它能产生强而持久的体液和细胞介导的 免疫应答（CMI）是指对在其基因组中表达的异源抗原的免疫应答。我们最近生成了 具有内置安全机制的VACV载体，仅在四环素存在下复制 抗生素当作为疫苗施用时（在不存在抗生素的情况下），载体不复制 但保留了其免疫原性，因此对人使用更安全。方便地，载体可以是 在四环素存在下以高滴度在细胞培养中繁殖，不像其他复制缺陷型 基于VACV的载体，例如修饰的安卡拉牛痘（MVA）。最近，我们开发了一种新的 本发明提供了一种在不使用选择标记的情况下产生和纯化重组VACV疫苗的方法， 短短一周，而这个过程通常需要几个月。我们的目标是用这种方法迅速 产生表达ZIKV E和M蛋白的重组VACV疫苗，并测试它们的 在小鼠中的免疫原性、安全性和功效。在目标1中，我们将快速生成和表征 表达ZIKV E蛋白的复制缺陷型VACV疫苗候选物（VACV-ZIKV）。一些 ZIKV E和M-E基因构建体设计用于单独表达E或作为病毒样颗粒表达 在一些实施方案中，使用VLP以最大化血清中和（SN）抗体的诱导。在目标2中，我们将测试 图1显示了VACV-ZIKV疫苗候选物在小鼠中的免疫原性（SN滴度和CMI应答）和安全性。 同时，在Aim 3中，我们将基于当前模型开发ZIKV小鼠激发模型， 相关DENV来测试我们的VACV-ZIKV候选疫苗的功效。我们的目标是利用我们的 加速VACV平台，以快速选择导致高水平SN的最佳抗原策略 抗体产生、CMI应答和小鼠保护。

PROJECT SUMMARY Zika virus (ZIKV) is an enveloped flavivirus transmitted by Aedes mosquitos which leads to an asymptomatic or mild dengue-like disease. A large ZIKV outbreak started in Brazil in early 2015, recently spreading to more than 25 countries in the Americas. Alarmingly, the ZIKV Brazilian outbreak has been linked to thousands of microcephaly cases in babies born to infected mothers, leading the WHO to recently declare a Public Health Emergency of International Concern. Thus, control efforts are desperately needed to contain the outbreaks and avoid further spread to other countries. No ZIKV vaccines have been developed, although vaccines for the related dengue virus (DENV) based on the Membrane (M) and Envelope (E) proteins, which induce protective immunity, are in advanced clinical trials, and one (Dengvaxia) was just approved in Mexico, Brazil, and the Philippines. Vaccinia virus (VACV) was used to eradicate smallpox, a disease caused by variola virus, a related poxvirus. VACV has also been successfully used as a replication-competent or -defective viral vector for the development of effective human and animal vaccines, as it elicits strong and long-lasting humoral and cell-mediated immune (CMI) responses to heterologous antigens expressed in its genome. We recently generated VACV vectors with a built-in safety mechanism that replicate only in the presence of tetracycline antibiotics. When administered as a vaccine (in the absence of antibiotics), the vector does not replicate but retains its immunogenicity, and therefore is safer for human use. Conveniently, the vector can be propagated in cell culture at high titers in the presence of tetracyclines, unlike other replication-defective VACV-based vectors such as modified vaccinia Ankara (MVA). More recently, we developed a novel method to generate and purify recombinant VACV vaccines without the use of selection markers in as little as one week, a process that normally takes months. Our goal is to use this method to rapidly generate recombinant VACV vaccines expressing the ZIKV E and M proteins and to test their immunogenicity, safety, and efficacy in mice. In Aim 1 we will rapidly generate and characterize replication-defective VACV vaccine candidates expressing the ZIKV E protein (VACV-ZIKVs). A number of ZIKV E and M-E gene constructs were designed for expression of E alone or as virus-like particles (VLPs) to maximize induction of serum neutralizing (SN) antibodies. In Aim 2 we will test the immunogenicity (SN titers and CMI responses) and safety of VACV-ZIKV vaccine candidates in mice. Concurrently, in Aim 3 we will develop a ZIKV mouse challenge model based on current models for the related DENV to test the efficacy of our VACV-ZIKV vaccine candidates. Our goal is to use our accelerated VACV platform to rapidly select the best antigen strategy that leads to high levels of SN antibody production, CMI responses, and protection in mice.