Preliminary Study On Human Monoclonal Antibodies Against The Glycoprotein Of Severe Fever With Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome is a new tick-borne infectious disease caused by Severe fever with thrombocytopenia syndrome virus.The disease has a high mortality rate and a risk of interpersonal transmission,however,there is still no specific means of prevention or treatment.Therefore,the development of effective and safe antiviral drugs has become an urgent need in the clinical management of Severe fever with thrombocytopenia syndrome.Severe fever with thrombocytopenia syndrome virus infection induces Severe fever with thrombocytopenia syndrome.The glycoprotein is an important surface protein of Severe fever with thrombocytopenia syndrome virus that forms a protrusion or spine on the viral envelope,which is cleaved to form two mature proteins,Gn and Gc.The Severe fever with thrombocytopenia syndrome virus glycoprotein plays an important role in the entry phase of the virus and is the mainly target for neutralization antibodies.Antibody drugs are currently being developed with the Severe fever with thrombocytopenia syndrome virus Gn as the main target.Therefore,the aim of this study is to isolate human monoclonal antibodies that can neutralize Severe fever with thrombocytopenia syndrome virus from patients recovering from Severe fever with thrombocytopenia syndrome,using the Severe fever with thrombocytopenia syndrome virus glycoprotein Gn as a bait antigen,by flow sorting techniques and single cell sequencing and VDJ sequencing,with the aim of rapidly developing therapeutic monoclonal antibodies against Severe fever with thrombocytopenia syndrome virus.From an antigenic perspective,the epitope peptide was obtained by comprehensive predictive analysis of the Severe fever with thrombocytopenia syndrome virus M fragment using bioinformatics techniques.The antigenic epitope peptide was synthesized and immunized into the mice,and the immunogenicity of the peptide was verified by ELISA and neutralization test.Candidate antigenic epitopes pave the way for the study of neutralizing antibodies in Severe fever with thrombocytopenia Syndrome.The main studies include:1.Preparation and activity evaluation of antibodies against the Severe fever with thrombocytopenia syndrome virus Gn: The Severe fever with thrombocytopenia syndrome virus Gn is known to play an important role in the process of virus infection.In this study,the Severe fever with thrombocytopenia syndrome virus glycoprotein Gn gene was synthesized by literature review and NCBI library comparison,the c DNA sequences were then cloned into the expression vector p ET-28 a and the expression vector p TT5 for recombinant expression of Gn in prokaryotic(E.coli)and eukaryotic expression systems(293F cell),respectively.The optimal induction temperature and time of expression of the recombinant protein were explored.The truncated and full-length proteins of Gn were successfully expressed.An indirect ELISA method for the detection of antibodies against Severe fever with thrombocytopenia syndrome was developed based on the Gn truncator expressed in the prokaryotic expression system.Patients recovered from Severe fever with thrombocytopenia syndrome within the last six months were subsequently contacted through the Jinan Centre for Disease Control and Prevention,their serum antibodies were tested and peripheral blood mononuclear cells were isolated from recovered patients who were seropositive.The isolated peripheral blood mononuclear cells were incubated with antigenic protein Gn with 6×His tag,Rb m Ab to 6×His tag(PE)and Ms m Ab to CD19(APC),and doublepositive cells were flow-sorted for single-cell sequencing and analysis of the results.The recombinant antibodies were tested for their binding and neutralization activity against SFTSV Gn.The results showed that the 22 recombinant antibodies did not show neutralization activity in the virus neutralization assay and all showed good binding activity when tested by ELISA.This suggests that the obtained recombinant antibodies can be used as detection antibodies for antigen detection.2.Screening and activity evaluation of Severe fever with thrombocytopenia syndrome virus glycoprotein epitope peptides: A total of 61 peptides were synthesised using bioinformatics techniques for the prediction of linear B-cell epitope polypeptides of Severe fever with thrombocytopenia syndrome virus M,as well as for the prediction of epitope polypeptides of Severe fever with thrombocytopenia syndrome virus L and S fragments as controls,and for the synthesis of peptides from published articles and patents.A mixture of the peptides with Freund’s adjuvant was used as the experimental group and a mixture of saline and adjuvant as the control.Mice were immunized five times by subcutaneous injection and serum was collected for binding and neutralization activity.Twenty-four of the predicted epitopes stimulated the production of polyclonal antibodies with binding activity;seven epitope peptides stimulated the production of polyclonal antibodies with neutralizing activity,which can be optimized and studied subsequently.In summary,this study expressed the Gn protein of Severe fever with thrombocytopenia syndrome virus and established an antibody detection method;the recombinant antibodies with good binding activity were isolated from recovered patients;antigenic epitopes of the Severe fever with thrombocytopenia syndrome virus were predicted and those with good immunogenicity could be used as candidates for the developing neutralization antibodies.