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）与严重的发病率和死亡率相关，由于其高传播率，其社会影响非常大。虽然目前存在针对SARS-CoV-2病毒株的有效疫苗，但预防性方法仍然有限，而且新的变异可能会限制现有疫苗的效力。刺突蛋白(S)是COVID-19的主要生物标志物，从病毒表面突出，促进病毒进入宿主细胞（例如肺部细胞），并诱导产生中和抗体。不幸的是，关于接种疫苗和感染者体内产生的SARS-CoV-2抗体水平和寿命的数据仍然有限。目前的技术价格昂贵，需要使用活病毒，并且不能定量监测个体的抗体水平。该项目的目标是探索一种新方法的可行性，该方法侧重于S上能够引发免疫反应并与产生的抗体结合以对抗该反应的区域（称为表位）。这些表位与COVID-19暴露产生的抗体之间的关系将使用研究人员开发的新型显示技术进行实时监测。该项目的结果将为代表活的SARS-CoV-2的肽和其特异性抗体的临床定量结合奠定基础。该项目有潜力利用合成生物学改进生物传感器工具，并将其扩展到其他病毒抗体、其他宿主的新感染暴发或生物威胁。该项目将为本科生和研究生提供培训机会，用于强调STEM/跨学科研究的重要性，重点是接触代表性不足的个人，并将保留一个关于新采用的研究策略的教学网络学习模块。该项目的长期目标是提高COVID-19抗体的快速检测和监测水平，提高疫苗的有效性。疫情爆发近三年来，人们对先前感染或接种疫苗的人群中与covid -19相关的保护性抗体的水平和寿命知之甚少。SARS-CoV-1和SARS-CoV-2共有的结构尖峰糖蛋白(S)通过介导宿主细胞进入和诱导产生中和抗体发挥关键作用，并被用于开发疫苗。对sars - cov感染中宿主s相关抗体的定量监测可以提供关键信息，但这种方法需要侧流试验（LFA）检测，这种方法昂贵且不够敏感。因此，需要可替代的、负担得起的、准确的COVID-19抗体（Abs）监测方法。为了解决这一迫切需求，该项目将探索可行性，使用S表位肽与进化RNA噬菌体Qβ的次要外壳蛋白A1融合的组合作为生物传感器开发的试剂。噬菌体Qβ很容易被规模化，是利未病毒科的一员，它通过F+菌毛感染细菌，并且具有二十面体外壳，可以抵抗极端条件。噬菌体Qβ是一种直径为25 nm的小正链RNA病毒，基因组长度为4.2 kb，编码4种蛋白质。它们是外壳蛋白（Cp），成熟蛋白（或A2），通读蛋白（A1）和RNA复制酶（RdRp）。最近，在研究者的实验室中，A1蛋白被成功地用于多肽的融合和暴露，这是重要的，因为它在Qβ平台的二十面体结构上的数量和位置。单独暴露在噬菌体平台上的sars - cov蛋白S表位将模仿其自然位置，识别并结合与换能器（链球菌II标签）强度降低成比例的抗体。以下三个目标将用于确定S表位探针和换能器肽在相互作用中的位置和作用，从而在生物测定中产生可检测和可测量的信号：(1)在A1蛋白末端构建和表达与换能器肽融合的S表位；(2)优化和稳定抗S抗体的S表位和传感器融合肽；(3)启动组装并表征生物传感器。在竞争性ELISA中，获得的重组噬菌体将根据市售抗体进行分析和标准化，并将用于实时检测和监测covid -19相关抗体。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值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.