Sentinel: RNA phage Qbeta displayed SARS-CoV-2 epitopes fused with Nanotag as a biosensor toolkit

严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)具有很高的发病率和死亡率，其高传播率使其具有很高的社会影响力。虽然目前存在针对SARS-CoV-2毒株的有效疫苗，但预防方法仍然有限，新的变种可能会限制当前疫苗的效力。新冠肺炎的主要生物标志物--刺突蛋白(S)从病毒表面投射出来，促进病毒进入宿主细胞(如肺部细胞)，并诱导产生中和抗体。不幸的是，关于接种疫苗和感染者产生的SARS-CoV-2抗体水平和寿命的数据仍然有限。目前的技术昂贵，需要使用活病毒，而且不是为了定量监测个体的抗体水平而设计的。这个项目的目标是探索一种新方法的可行性，这种方法专注于S身上能够引发免疫反应的区域(称为表位)，并与产生的抗体相结合来对抗这种反应。然后，将使用研究人员开发的一种新的显示技术，实时监测这些表位和接触新冠肺炎产生的抗体之间的关系。该项目的结果将为代表活体SARS-CoV-2的多肽及其结合为临床量化其特异性抗体奠定基础。该项目有可能用合成生物学改进生物传感器工具，并扩展到其他病毒抗体、其他宿主中新的感染暴发或生物治疗。该项目将为本科生和研究生提供培训机会，用于突出STEM/跨学科研究的重要性，重点是接触代表性较低的个人，并将保持一个关于新采用的研究策略的基于网络的教学学习模块。该项目的长期目标是改进对新冠肺炎抗体和疫苗效力的快速检测和监测。疫情爆发近三年后，人们对先前感染或接种疫苗的人群中保护性新冠肺炎相关抗体的水平和寿命知之甚少。结构尖峰蛋白(S)是SARS-CoV-1和SARS-CoV-2共有的糖蛋白，通过介导宿主细胞进入和诱导中和抗体的产生而发挥关键作用，并被用于疫苗的研制。在SARS冠状病毒感染中对宿主S相关抗体进行定量监测可以提供关键信息，但这种方法需要侧向流动分析(LFA)检测，这一方法昂贵且不总是足够敏感。因此，需要价格合理、准确的新冠肺炎抗体(Abs)监测方法。为了满足这一迫切需求，该项目将探索使用S表位多肽与进化RNA噬菌体Qβ的次要外壳蛋白A1融合作为生物传感器开发试剂的可行性。噬菌体Qβ很容易结垢，是Leviviridae家族的成员，它用F+菌毛感染细菌，并用二十面体外壳抵抗极端条件。噬菌体Qβ是一种直径为25 nm的小正链病毒，基因组大小为4.2kb，编码4种蛋白质。它们是外壳蛋白(CP)、成熟蛋白(或A2)、通读(A1)和RNA复制酶(RdRp)。最近，A1蛋白在研究者的实验室中成功地用于多肽的融合和表达，这一点很重要，因为它在Qβ平台的二十面体结构上的数量和位置。在噬菌体平台上分别暴露的SARS-CoV的蛋白S表位(S)将模拟它们的自然位置，识别并结合与转导(Strep II Tag)强度成比例的抗体。以下三个目标将用于确定S表位探针和转导子肽在生物检测中产生可检测和可测量信号的相互作用中的位置和作用：(1)构建和表达S表位与A1蛋白末端的转导子多肽的融合；(2)优化和稳定S表位和转导子融合多肽，以对抗S抗体；(3)启动组装和鉴定生物传感器。在竞争性酶联免疫吸附试验中，获得的重组噬菌体将被分析并与商业上可用的抗体进行标准化，并将被用于实时检测和监测新冠肺炎相关抗体。该奖项反映了美国国家科学基金会的法定使命，并已通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with significant morbidity and mortality, and its societal impact is very high due to its high rate of transmission. While effective vaccines exist for the current strains of SARS-CoV-2, preventative approaches remain limited, and novel variants may limit the efficacy of current vaccines. The spike protein (S), the major biomarker of COVID-19, projects from the surface of the virus, facilitates the virus’s entry into host cells (e.g. cells that line the lungs), and induces the production of neutralizing antibodies. Unfortunately, data on the level and longevity of SARS-CoV-2 antibodies produced in vaccinated and infected people are still limited. Current technologies are expensive, require the use of live viruses, and are not designed to quantitively monitor the level of antibodies in individuals. The goal of this project is to explore the feasibility of a new approach that focuses on the regions on S (called epitopes) that are capable of eliciting an immune response and of combining with the antibody produced to counter that response. The relationship between these epitopes and antibodies resulting from COVID-19 exposure will then be monitored in real-time using a novel display technology that has been developed by the investigators. Results from this project will create a foundation for peptides representing live SARS-CoV-2 and binding for clinical quantification of its specific antibodies. The project has the potential to improve biosensor tools with synthetic biology and to expand to other viral antibodies, new infection outbreaks in other hosts, or biothreats. The project will provide training opportunities for undergraduate and graduate students, be used to highlight the importance of STEM/interdisciplinary research, with an emphasis on reaching out to underrepresented individuals, and will maintain a pedagogical web-based learning module about the newly adopted research strategies.The long-term goal of this project is to improve the rapid detection and monitoring of COVID-19 antibodies and vaccine efficacy. Nearly three years into the pandemic, very little is known about the level and longevity of protective COVID-19-related antibodies in previously infected or vaccinated populations. The structural spike (S) glycoprotein, common to both SARS-CoV-1 and SARS-CoV-2, plays a pivotal role by mediating the host cell entry and inducing the production of neutralizing antibodies, and was used to develop the vaccines. A quantitative monitoring of host S-related antibodies in SARS-CoVs infections could provide key information, but this method requires lateral flow assay (LFA) testing, which is expensive and not always sensitive enough. Thus, alternative affordable and accurate COVID-19 antibodies (Abs) monitoring methods are required. To address this urgent need, the project will explore the feasibility, using a combination of S epitope peptides fused with the minor coat protein A1 of the evolutionary RNA phage Qβ displayed as reagents for biosensors development. Phage Qβ is easily scaled and is a member of the family Leviviridae that infects bacteria with the F+ pilus and is resistant to extreme conditions with an icosahedral shell. Phage Qβ is a small positive strand RNA virus 25 nm in diameter and a 4.2 kb genome encoding 4 proteins. These are coat protein (Cp), maturation (or A2) protein, read-through (A1), and the RNA replicase (RdRp). Recently, the A1 protein was successfully used for fusion and exposition of peptides in the investigator’s laboratory, which is important due to its number and positions on the icosahedral structure of the Qβ platform. Protein S epitope(s) of SARS-CoVs separately exposed on the phage platform will mimic their natural position, recognize, and bind to an antibody proportional to the reduction of the transducer’s (Strep II tag) intensity. The following three objectives will be used to determine the position and role of the S epitope probes and the transducer peptide in an interaction that leads to a detectable and measurable signal in a bioassay: (1)To construct and express S epitopes fused with transducer peptide at the end of A1 protein; (2) To optimize and stabilize the S epitopes and transducer fusion peptides against anti-S antibodies; and (3) To initiate the assemblage and characterize the biosensor. In a competitive ELISA, the recombinant phages obtained will be analyzed and standardized against commercially available antibodies and will be used to detect and monitor COVID-19-related antibodies in real time.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.