Alain Bopda Waffo
Alain Bopda Waffo: Associate Professor of Biochemistry and Molecular Biology.
Department of Biochemistry and Molecular Biology
RNA phage Qbeta engineered as biosensor for coronavirus diseases point-of-care
The world is still facing the health and economic damage due to the coronavirus pandemic and the enormous challenges of laboratory testing. Among the coronavirus reports so far, SARS-CoV-1 and SARS-CoV-2 are related, highly contagious and virulent, and cause the severe acute respiratory syndromes (SARS). The common spike (S) glycoprotein plays a pivotal role, mediates the host cell entry, and induces neutralizing antibodies. The S protein can be assigned the role of biomarker for SARS-CoVs infections and zoonoses. Detecting and monitoring coronaviruses in symptomatic and asymptomatic infected people require sophisticated analytical techniques that combine serology and genome testing, which are expensive. Serological testing uses to determine protective antibodies from vaccination is time- and labor-intensive process and can require the live virus or pseudo-virus. Consequently, alternative detection and monitoring methods are urgently needed. The use of naturally occurring phages has flourished in the field of synthetic biology and can be used as biosensors for biorecognition of a wide range of probes to achieve high degree of sensitivity and specificity. Initially, we will endeavor to engineer RNA Qβ phage to map and elucidate the S protein domains recognizing antibody and angiotensin-converting enzyme 2, lipoprotein and related host cell receptors (DPP4, CD147). Additionally, we propose to explore the feasibility, using a combination of S epitope peptides with the minor coat protein A1 of the RNA phage Qβ displayed as reagents for biosensors. We will fuse and present separately, the epitopes of the spike protein of both coronavirus on the RNA phage Qβ display platform together with a transducer streptavidin tag peptide. The hybrid phage obtained will be used to concentrate, detect, and monitor in real time the SARS antibodies in human and other hosts given the evolution and zoonosis of coronaviruses infection. Previously, phage engineering was mostly applied to M13 DNA phage, but DNA phages prevent any rapid evolution and adaptation and display at only one of its filamentous structure ends. RNA phages possess a replicase that lacks the proofreading activity which, fuels and contributes to the evolution and adaptation, allowing for optimization of engineering efforts. Phage Qβ is easily scaled and is a member of Leviviridae that infects bacteria with the F+ pilus found in wastewater and resistant to extreme conditions. Phage Qβ is a small positive strand RNA virus with a 25 nm diameter and a 4.2 kb genome encoding 4 proteins. These are coat protein (Cp), maturation (or A2) protein, read-through or minor coat protein (or A1), and the RNA-dependent RNA polymerase (RdRp) or RNA replicase. Recently, the A1 protein has been successfully used for fusion and exposition of peptides in our laboratory, which is important for its number and position on the icosahedral structure of Qβ platform. The Qβ displaying A1-S can mimic the SARS-CoVs’ and be efficiently used as a point of care for SARS disease. These concepts and findings, with intricate molecular engines, could provide an improved an innovative tool in biosensing. Additionally, this project will provide interdisciplinary training to students at the interface of bioinformatics, biophysics, and biology. We have acquired most of the materials for this study. The long-term goal of this project is to improve the rapid detection and monitoring of SARS in the United States. To reach this goal, results from this project will create a foundation for peptides representing live SARS-CoV-1 and SARS-CoV-2 and binding their specific antibodies. By utilizing randomized peptide(s) the project has the potential to expand to other RNA viruses (EBOV, ZIKV, and HIV), new infection outbreak other hosts, and biothreats and explosive chemicals and/or precursors and to improve biosensor with synthetic biology.