Abstract
BackgroundSFTS virus (SFTSV) is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS) in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection.Methodology/Principal FindingsWe have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs) recognized the nucleocapsid (N) protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs.Conclusions/SignificanceThe large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.
Highlights
Severe fever with thrombocytopenia syndrome (SFTS), with average case fatality rate of 12%, is an emerging infectious disease caused by a newly discovered virus, named severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) [1]
Conclusions/Significance: The large number of human recombinant monoclonal antibodies (MAbs) derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTS virus (SFTSV) infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection
Using the pComb 3H vector system, the SFTSV patients derived light chain library was first constructed with a complexity of .1.06107 clones, the Fd heavy chain genes were efficiently inserted into the light library vectors and resulted in a initial combinatorial Fab library with a complexity of .1.06108 independent clones and 100% Fab genes diversity after sequencing confirmation
Summary
Severe fever with thrombocytopenia syndrome (SFTS), with average case fatality rate of 12%, is an emerging infectious disease caused by a newly discovered virus, named SFTS virus (SFTSV) [1]. The N protein of 245 amino acids, as in the case of many other nucleoproteins of negative strand RNA virus, acts as a scaffold for packing of virus. Antibodies against SFTSV N protein were commonly detected early after infection, which suggested the N protein is possible to be used as target antigen for early diagnosis of SFTSV infection. It has been reported that the viral N protein is highly immunogenic for many members in the genus of Phlebovirus and acts as major antigen [4,5]. We for the first time described the generation and characterization of a panel of human monoclonal antibodies (MAbs) against SFTSV N protein using phage display library approach, and the targeted antigenic epitopes were further mapped by competitive assay, molecular modeling and site-directed mutations. The results provided in this study could facilitate understanding of humoral responses to SFTSV infection and help to develop diagnostic tools for detection and diagnosis of SFTSV infection at various infection stages, which could be applied in clinical work as well as epidemic surveillance
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