Development of a highly sensitive serological assay for Covid-19 based on the use of virus-like particles and protein-based nanoparticles.

我们建议使用杆状病毒表达系统来产生模仿最近出现的SARS-CoV-2的病毒样颗粒(VLP)。我们先前利用我们实验室开发的单一杆状病毒表达载体共表达了SARS-CoV的三个基本结构蛋白(S、M和E)，并证明了当这三个蛋白在Sf9细胞中共表达时，这三个蛋白能够自发地采用模仿完整病毒的构象(1)。随后的小鼠研究表明，用这些疫苗接种会引起强烈的中和抗体反应，相当于在恢复期的人身上看到的反应。最近，我们利用这些已有的试剂作为基础来生产SARS-CoV-2 VLP，将S、M和E蛋白替换到先前构建的杆状病毒载体中，初步数据表明这三种蛋白都得到了表达。我们建议对表达的蛋白、VLP的形成和小鼠的免疫反应进行彻底的表征，以验证它们作为病毒模拟物的真实性。VLP最终将被用于开发抗体检测分析。同时，我们将表达SARS-CoV-2 Fc标记的S蛋白，并将其与我们已经制备的基于鲁马嗪合成酶蛋白的纳米颗粒(PNPs)外包裹。我们已经证明，对于不同病毒的受体结合蛋白，类似的纳米颗粒增强了结合亲和力和亲和力。这些PNPs自组装成中空单分散的球形结构，稳定了三聚体S蛋白并增强了抗原性(2)。SARS-CoV-2VLP和PNPs都是开发高灵敏度诊断方法的优秀候选者，该方法将用于检测新冠肺炎感染的人血清。

We propose to use the baculovirus expression system to generate Virus-Like Particles (VLPs) mimicking the recently emergent SARS-CoV-2. We have previously co-expressed three essential structural proteins (S, M, and E) of SARS-CoV using a single baculovirus expression vector developed in our lab and demonstrated that when co-expressed in Sf9 cells, these three proteins were able to spontaneously adopt a conformation mimicking the full virus (1). Subsequent mouse studies showed that vaccination with these elicited a strong neutralising antibody response, equivalent to that seen in a convalescent human. Recently we used these pre-existing reagents as a basis to produce SARS-CoV-2 VLPs, substituting the S, M, and E proteins into the previously generated Baculovirus constructs, and preliminary data shows that all three SARS-CoV-2 proteins are expressed. We propose to characterise thoroughly the expressed proteins, the formation of VLPs, and the immune response in mice to validate their authenticity as virus mimics. VLPs will be ultimately used to develop an antibody detection assay. In parallel, we will express the SARS-CoV-2 Fc-tagged S protein alone to be externally encapsulated with the lumazine synthase protein-based nanoparticles (PNPs) that we have already prepared. We have shown that similar nanoparticles for a receptor binding protein of a different virus enhanced binding affinity and avidity. These PNPs self-assemble into hollow monodisperse spherical structures, which stabilises the trimeric S protein and enhances antigenicity (2). Both SARS-CoV-2 VLPs and PNPs are excellent candidates for the development of highly sensitive diagnostic assay that will be used to detect COVID-19 infected human sera.