Generation And Characterization Of Human Neutralizing Antibodies Against Hantaan Virus

Hemorrhagic fever with renal syndrome（HFRS）caused by Hantavirus（HTV）is an acute infectious disease characterized by fever,hemorrhage and acute renal impairment.HTV is distributed worldwide,and Shaanxi province is one of the high incidence areas of HFRS in China.On the one hand,the HFRS vaccination program has been implemented in the population of key epidemic areas in China,the effectiveness of the vaccine against HFRS needs to be evaluated periodically.On the other hand,anti-Hantaan virus（HTNV）specific neutralizing antibodies（NAb）can be directly combined with HTNV to participate in the immune clearance of the virus and few human NAb has been used for emergency prevention and treatment of HFRS,it is necessary to prepare human antibodies with potential neutralizing activity.Firstly,we evaluated the sensitivity and specificity of two different kits.Then,we evaluated the immune efficacy of the HFRS vaccine.Finally,we constructed Phage Fab antibody library against HTNV with the NAb positive B lymphoblastoid cell lines（BLCLs）of the vaccine recipients and patients.Human anti-HTNV Fab antibodies with neutralizing activity were obtained,which may provide the approach for the emergency prevention,treatment and disease diagnosis of HFRS.To this end,we carried out the following experiments:Part 1:Establishment,screening and cloning of BLCLs of HFRS vaccine recipients and patientsFirst,the sensitivity and specificity of domestic and imported HFRS IgG antibody ELISA kits were evaluated respectively for HTNV antibody detection.The results showed that the sensitivity of the domestic kit is higher than that of the imported kit while the specificity of imported kit is better than that of the domestic kit.Then,blood samples of HFRS vaccinated persons and normal subjects were collected respectively and seventy BLCLs of vaccine recipients were established by Epstein-Barr virus（EBV）transformation technology.The HTNV specific antibodies in the serum samples and the supernatant of BLCLs were tested,and the immunization effect of HFRS vaccination in Xianyang area was evaluated.The level and positive rate of HTNV nucleocapsid protein（NP）specific IgM,IgG antibody and NAb were significantly increased in the sera of the vaccinated population,and the three groups had a good correlation.The most intense antibody secreting cell response occurred 3 months after vaccination,consistent with the results of the serum.The results showed that HFRS vaccine immunization program in the northwest of China’s population can effectively induce humoral immunity.Finally,NAb positive BLCLs were identified and BLCLs were cloned.Thirty-five NAb positive BLCLs of HFRS vaccinated persons and patients were used to establish a phage antibody library with potential neutralizing activity against HTNV.Successful cloning of BLCLs is expected to open up new way for monoclonal antibody（mAb）production.Part 2:Construction and screening of phage Fab antibody library against HTNVmRNA from NAb-positive BLCLs was extracted and cDNA was synthesized.Based on these,an anti-HTNV phage Fab antibody library with a capacity of 1.3×10⁹ was established by phage display technology.The positive insertion rate of Fab was 95%and the correct rate of antibody sequence was 68%.The HTNV Fab phage antibody was screened by solid phase screening method after 4 rounds of adsorption,elution and amplification using HTNV as antigen.The elution phage titer showed an increase in recovery.The correct rate of random clones was 68%before screening and 81%after the fourth round screening.The diversity was 95%before screening and 31%after the fourth round screening.Finally,fifteen specific HTNV phage Fab antibodies were selected from50 colonies in the third and fourth round screening.In conclusion,we successfully constructed and screened the anti-HTNV phage Fab antibody library.Part 3:Soluble expression and detection of neutralizing activity of human anti-HTNV Fab antibodyThe specific HTNV Fab phage obtained by screening was transfected into Ecoli.HB2151 to induce the expression of soluble HTNV Fab antibody and purified by affinity chromatography.The products of expression and purification were analyzed by reduction electrophoresis.It was found that the apparent band at about 25 kDa was consistent with the expected size of Fab.The enzyme linked immunosorbent assay（ELISA）indicated that these soluble HTNV Fab antibodies could specifically bind to HTNV.Neutralization experiments showed that three Fab antibodies exhibited neutralizing activity.Sequence analysis showed that these Fab antibodies belonged to 6different antibody families,which showed a family tendency.Most positive clones of the light chain and heavy chain belonged to the V3 family and V4 family.The family of IGKV3-20*01 and IGHV4-59*01 were the main ones.Homology analysis of nucleotide sequence and amino acid sequence showed that the gene sequences of 4-9Fab antibody and three other Fab antibodies with neutralizing activity came from human germline genes,and were different from the existed antibody gene sequences.Part 4:ER stress induced by HTNV in dTHP-1 cellsAlthough HTV infection could induce strong immune responses,the mechanisms on it still need to be further clarified.In this part,we explored whether HTNV infection could induce the ER stress in d THP-1 cells.It showed that the mRNA and protein levels of ER stress related protein78kDa glucose-regulated protein（GRP78）and mRNA level of X box-binding protein 1（XBP-1）after HTNV infection were significantly higher than those in uninfected control group.These results indicated that the HTNV infection could induce the ER stress in dTHP-1 cells.However the mRNA level of C/EBP homologous protein（CHOP）was not significantly different between the infected group and the untreated group in 2h after virus infection.Meanwhile dTHP-1 cells infected with HTNV at 12h did not show obvious apoptosis.These results suggested that HTNV infection did not directly lead to the apoptosis of d THP-1 cells during 12 h after the virus infection.