Rapid Generation of Vaccine Candidates Against Novel Coronavirus (SARS-CoV-2) Using the Bacteriophage T4 Nanoparticle Platform

项目总结 这项提议旨在迅速产生针对2019年新型冠状病毒SARS-CoV的候选疫苗。 2.自从大约三个月前出现以来，这种病毒已经造成了12万多人感染和4300人 全球范围内的死亡人数，并正在迅速蔓延到几乎所有国家，包括美国。这是全球性的 卫生紧急情况必须立即通过迅速发展的医疗对策加以解决。 我们的噬菌体(噬菌体)T4疫苗平台特别适合应对这一威胁。在PI中开发 实验室已经证明，T4疫苗可以产生强大的体液和细胞免疫反应 并在多种动物模型中提供对炭疽和鼠疫的完全保护，包括小鼠、大鼠、 兔子和猕猴。T4疫苗不需要佐剂，因为它的表面结构模仿病原体- 病毒病原体的相关分子模式(PAMP)，并刺激强大的先天和获得性免疫。 120x86 nm噬菌体T4衣壳包装有171kb的基因组，并装饰有两个非必需的 外衣壳蛋白；870个SOC(小外衣壳蛋白)分子和155个拷贝(高度抗原性 外衣壳蛋白)。在具体目标1中，将通过整合SARS构建一系列T4冠状噬菌体- 冠状病毒-2病毒粒子组件单独和组合，由CRISPR工程。编码该基因的 在CAG强启动子的控制下，整个穗外区将被插入到噬菌体基因组中。vt.在.的基础上 免疫后，宿主细胞(免疫部位的肌细胞和抗原提呈细胞)吞噬噬菌体 并持续分泌胞外区域三聚体，刺激免疫系统数周至数月。 S蛋白受体结合域的基因将被插入，以便表达该受体结合域 在宿主细胞中，以及在大肠杆菌中作为Soc融合蛋白，该融合蛋白将展示在高达870的噬菌体衣壳上 每个衣壳的拷贝数。E蛋白的胞外区将被融合到Royal上，每个衣壳最多显示155个拷贝。 最后，约400份N蛋白将被包装在衣壳内，作为支架核心的一部分。 在特定目的2中，将评估上述T4冠状重组噬菌体对SARS冠状病毒的诱导作用。 2小鼠模型中的病毒粒子特异性免疫反应。用纯化的噬菌体颗粒免疫小鼠 肌内和免疫反应将通过ELISA法、竞争性受体结合法、ELISpot、 病毒中和试验。我们预计T4冠状病毒疫苗将产生强大的抗体和细胞 这份报告还将告知哪位候选人(S)在阻断SARS-CoV-2感染方面最有效。 我们已经简化了CRISPR工程，以便拟议的T4疫苗可以在 大约4周，动物试验可以在大约12周内完成。候选疫苗将是 可用于临床试验和疫苗生产。T4疫苗将非常容易制造， 扩大规模，并在全球范围内分布，并有可能导致避免冠状病毒危机的突破。

PROJECT SUMMARY This proposal aims to rapidly generate vaccine candidates against the 2019 novel coronavirus SARS-CoV- 2. Since its emergence about three months ago, this virus has caused more than 120,000 infections and 4,300 deaths worldwide and is rapidly spreading to virtually every country including the United States. This global health emergency must be immediately addressed by rapidly developing medial countermeasures. Our bacteriophage (phage) T4 vaccine platform is uniquely suited to address this threat. Developed in PI's laboratory, the T4 vaccines have been proven to generate robust humoral as well as cellular immune responses and confer complete protection against anthrax and plague in multiple animal models including mice, rats, rabbits, and macaques. The T4 vaccines do not need an adjuvant as its surface structure mimics the Pathogen- Associated Molecular Patterns (PAMPs) of viral pathogens and stimulate strong innate and adaptive immunity. The 120 x 86 nm phage T4 capsid is packaged with 171 kb genome and decorated with two non-essential outer capsid proteins; 870 molecules of Soc (small outer capsid protein) and 155 copies of Hoc (highly antigenic outer capsid protein). In specific aim 1, a series of T4-corona phages will be constructed by incorporating SARS- CoV-2 virion components individually and in combinations, by CRISPR engineering. The gene encoding the entire spike ectodomain will be inserted into phage genome under the control of the strong CAG promoter. Upon immunization, host cells (myocytes and antigen presenting cells at the site of immunization) take up phage particles and secrete the ectodomain trimers continuously, stimulating the immune system for weeks to months. The gene for the receptor binding domain (RBD) of S protein will be inserted such that the RBD will be expressed in host cells, as well as in E. coli as a Soc fusion protein which will then be displayed on phage capsid up to 870 copies per capsid. The ectodomain of E protein will be fused to Hoc and displayed up to 155 copies per capsid. Finally, ~400 copies of N protein will be packaged inside the capsid as part of the scaffolding core. In specific aim 2, the above T4-corona recombinant phages will be evaluated for elicitation of SARS-CoV- 2 virion-specific immune responses in a mouse model. Mice will be immunized with purified phage particles intramuscularly and the immune responses will be quantified by ELISA, competitive receptor binding, ELISpot, and virus neutralization assays. We expect that the T4-corona vaccines will elicit robust antibody and cellular responses and also inform which candidate(s) will be most effective in blocking SARS-CoV-2 infection. We have streamlined the CRISPR engineering such that the proposed T4 vaccines can be constructed in about 4 weeks and the animal testing can be completed in about 12 weeks. The candidate vaccines will then be available for clinical trials and vaccine manufacture. The T4 vaccine will be exceedingly easy to manufacture, scale, and distribute globally, and could potentially lead to a breakthrough to avert the coronavirus crisis.