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

项目摘要 该提案旨在快速产生针对2019年新型冠状病毒SARS-CoV的候选疫苗- 2.自大约三个月前出现以来，这种病毒已造成超过12万人感染， 死亡人数在世界范围内迅速蔓延到几乎所有国家，包括美国。这一全球 必须通过迅速制定医疗对策来立即处理卫生紧急情况。 我们的噬菌体（噬菌体）T4疫苗平台非常适合解决这一威胁。在PI中开发 在实验室中，T4疫苗已被证明能产生强大的体液和细胞免疫应答 并在包括小鼠，大鼠， 兔子和猕猴T4疫苗不需要佐剂，因为其表面结构模拟病原体- 相关的分子模式（PAMPs）的病毒病原体和刺激强大的先天性和适应性免疫。 120 × 86 nm噬菌体T4衣壳包装有171 kb基因组，并用两个非必需的 外衣壳蛋白; 870分子的Soc（小外衣壳蛋白）和155拷贝的Hoc（高抗原性 外衣壳蛋白）。在具体目标1中，将通过并入SARS- CoV-2病毒体组分单独和组合，通过CRISPR工程。编码基因 整个刺突胞外域将在强CAG启动子的控制下插入噬菌体基因组中。后 免疫时，宿主细胞（免疫部位的肌细胞和抗原呈递细胞）摄取噬菌体 颗粒并连续分泌胞外域三聚体，刺激免疫系统数周至数月。 将插入S蛋白的受体结合结构域（RBD）的基因，使得RBD将被表达 在宿主细胞中，以及在E.大肠杆菌作为Soc融合蛋白，然后将其展示在噬菌体衣壳上高达870 拷贝数。E蛋白的胞外域将与Hoc融合，每个衣壳展示多达155个拷贝。 最后，约400个拷贝的N蛋白将被包装在衣壳内作为支架核心的一部分。 在具体目标2中，将评价上述T4-冠状病毒重组质粒的SARS-CoV诱导。 小鼠模型中的2种病毒体特异性免疫应答。将用纯化的噬菌体颗粒免疫小鼠 通过ELISA、竞争性受体结合，ELISpot， 和病毒中和试验。我们期望T4冠状病毒疫苗将引发强大的抗体和细胞免疫。 反应，并告知哪些候选人将最有效地阻断SARS-CoV-2感染。 我们已经简化了CRISPR工程，使得拟议的T4疫苗可以在 约4周，动物试验约12周完成。候选疫苗将在 可用于临床试验和疫苗生产。T4疫苗将非常容易制造， 规模，并在全球范围内分布，并可能导致突破，以避免冠状病毒危机。

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.