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

点击翻译按钮获取中文摘要

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.