A Robust Reporting System For Measuring SARS-CoV-2 Spike-Mediated Cell-Fusion Efficiency & SARS-CoV-2 MRNA Vaccine Research

The emergence and rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2(SARS-CoV-2)is disrupting global health and economies.SARS-CoV-2 infects host cells mainly through membrane fusion mediated by-viral spike protein and human ACE2 protein.Secondly,the spike protein can also mediate cell-cell fusion,and its cell-fusion ability is related to the infectivity and spread of the virus.Therefore,monitoring the cell-fusion efficiency mediated by the spike protein of SARS-CoV-2 is very important for the prevention,control and treatment of the virus.In addition,the continuous emergence and iteration of SARS-CoV-2 variants has greatly affected the protection efficiency of currently marketed vaccines.Therefore,the design and development of efficient and broad-spectrum SARS-CoV-2 vaccines is still the key to future epidemic prevention and control.This research mainly includes the following two parts:Part I:An intuitive,visual,and quantifiable reporting system for cell fusion was established.The specific research contents are as follows:(1)Design and complementarity analysis of cell-fusion reporter system.Plasmids CGFL,LFCG,NGJS,SJNG were constructed by fusing split m Neon Green fluorescent protein and split Nano Luc luciferase at both ends of a pair of interacting parallel leucine zippers(b Fos and b Jun).The combination of NGJS and CGFL produced the optimum results by co-transfection to achieve the expression of m Neon Green green fluorescence signal and Nano Luc luciferase activity.And it was found that when the luciferase activity detection value was in the range of 1.0×10~3～1.0×10~6,there was a good linear relationship.(2)Feasibility analysis of cell-fusion reporting system.The plasmids of ACE2 and NGJS were co-transfected into HEK293T cells as target cells,and the spike plasmid of D614G mutant strain and CGFL were co-transfected as effector cells.After co-culturing the two kinds of cells,cell fusion was observed by fluorescence microscope,and the luciferase activity was monitored.The results showed that the system can be used to evaluate the cell-fusion efficiency mediated by SARS-CoV-2 spike.(3)Application of cell-fusion reporting system.The cell-fusion efficiency of SARS-CoV-2 Variant of Concern(VOC)spikes was monitored using this system,and the cell-fusion efficiency results showed that:Delta variant>Alpha variant≈Beta variant>D614G variant>Omicron variant.Using this detection system,the effects of two clinical-used neutralizing antibodies REGN10933 and REGN10987 on cell fusion efficiency were evaluated.Both antibodies and their combinations were found to significantly inhibit Delta spike-mediated cell-fusion,while the efficiency of Omicron spike-mediated cell fusion was not affected by either antibody,suggesting that the two neutralizing antibodies not only leads to loss Omicron infection-suppressing activity,but also to lose the ability to inhibit Omicron spike-mediated cell-fusion.Part II:Two trimer mRNA vaccines were designed and preliminarily evaluated based on the receptor binding domain(RBD)of SARS-CoV-2Delta spike protein.The specific research contents are as follows:(1)Design and optimization of SARS-CoV-2 spike protein RBD trimer mRNA vaccine.The signal peptide sequence and the number of glycosylation sites in the RBD region were optimized respectively,and the optimal sequences were screened.Finally,two candidate RBD-trimer sequences were selected to mimic the natural conformation of SARS-CoV-2 spike trimer for animal research.mRNA was synthesized by in vitro transcription and encapsulated by SM102 cationic using solvent injection way.(2)The two RBD-trimer mRNA vaccines were used to immunize mice at a dose of 10μg/mouse,and the results showed that sequence 1 could rapidly induce efficient neutralizing antibody production in mice after the first immunization.After the second immunization,the level of neutralizing antibodies induced by the two sequence in vivo was significantly increased,and long-acting and broad-spectrum effects were induced.Besides,Th1-type cellular immune responses in vivo could be induced by both vaccines.In general,the sequence 1 is better than the sequence 2 and can be used for further research.In conclusion,a cell-fusion reporting system was firstly constructed in this study,which can conveniently and quickly detect the cell-fusion efficiency mediated by SARS-CoV-2 spike.It has the advantages of simple and time-saving operation,visualization of results,and simultaneous qualitative and quantitative analysis,providing a new and reliable technical method for quickly judging the fusion activity of known or unknown variants of SARS-CoV-2 spikes.Secondly,two RBD-trimer mRNA vaccines were designed and preliminarily evaluated.Results showed that high,long-term,and broad-spectrum neutralized antibodies were induced after two immunizations in mice,which would be worthy for further research.