Tick-borne Encephalitis Virus Neutralizing Antibody Research

Tick-borne encephalitis(TBE),or called forest encephalitis,transmitted to human by ticks,could cause severe or fatal infection of the central nervous system.The pathogen of this disease is the tick-borne encephalitis virus(TBEV).According to the World Health Organization(WHO)statistics,more than 10000 TBE cases per year were reported in the world.In China,TBE is classified as one of the five major occupational infectious diseases.There was a dramatic increase in TBE cases in recent years.This increase may be due to the increasing number of people entering the forest area with the rapid development of tourism and adventure activities.In addition,tick-borne encephalitis virus is one of the important biological warfare agents,the United States CDC has classified it as virus of class C biological weapons.There is currently no specific treatment available for TBE,which has high mortality and morbidity.Symptomatic treatment consists of supportive and antiserum therapy clinically.Vaccination is one of the primary means of preventing TBE infection.There is still no report on research of fully human antibodies against TBEV recently,most of the reported ones were mouse-derived monoclonal antibodies used in laboratory.Thus the purpose of our research is to provide a new available treatment with fully human antibodies against TBEV,which are prepared based on the fluorescence cell sorting and single cell PCR technique.In this study,after signed informed consent,2 male volunteers(25-40 years old)were immunized with TBEV inactivated vaccine at day 0,14,and boosted at day 42.Blood samples were collected at day 0,21,28,49,and 63.TBEV specific neutralizing antibody serum titers at different time point were detected by ELISA.The peripheral blood mononuclear cells(PBMCs)were isolated from blood sample at day 49 using Ficoll-hypaque method.After fluorescent labeling,specific antibody secreting cells(ASCs)with CD19+/CD3-/CD20-/CD27high/CD38 high were isolated from PBMCs.1632 of single ASCs was isolated,accounting for 0.02% of the total number of cells.Among these,349 pairs of monoclonal antibody genes were amplified using RT-PCR and nested PCR,the amplification efficiency was about 21.38%.At last,175 m Abs were left after sequencing and analyzing by IMGT/V-QUEST.The linear expression cassette of antibody genes was constructed using overlapping PCR,and transfected into Expi 293 F cells.14 TBEV specific binding monoclonal antibodies were found by ELISA.Of the 14 m Abs,2 had capacity of neutralizing TBEV in BHK-21 cells(1C4 and 3E12).1C4 and 3E12 m Abs could play a complete protective effect on cells with antibody concentration≥200 μg/m L.1C4 has a better protective effect(IC50=75 μg/m L)than 3E12(IC50=108 μg/m L).TBEV is a member of the genus Flavivirus,family Flaviviridae.It has a positive sense,single stranded RNA genome.The genes of protein E and NS1 were cloned into p ET-32 a vector.Then the correct recombinant plasmid was expressed in E.coli with IPTG induction and purified by Ni-NATB bead.The interaction of antigens and antibodies was tested by ELISA and Western Blot,and found that the positive serum only reacted with protein E.This result suggested that the active ingredient of vaccine used in our study was protein E.Neutralizing antibodies 1C4 and 3E12 have weak binding activity with protein E.Considering the lack of glycosylation of antigenic proteins expressed in prokaryotic,and TBEV membrane protein Pr M also plays an important role in the process of viral replication and maturation,we cloned the genes of protein Pr ME and NS1 into p DC316 vector.The correct recombinant plasmid was expressed in 293 T cells and identified by Western Blot with positive serum.The results were consistent with expression in prokaryotic.We found that in the Western Blot reaction of protein E with antibodies,there were no positive results when antibody used alone;but when antibodies mixed together,a positive purpose band appeared.These results suggested that the monoclonal antibodies we screened bind to the different epitopes of the antigen E protein.In summary,2 TBEV specific neutralizing antibodies were screened based on the fluorescence cell sorting and single cell PCR technique.Recombinant protein E and NS1 were expressed successfully in E.coli and 293 T cells.Neutralizing antibodies have weak binding activity to protein E.