Generation And Characterization Of Cross-reactive Monoclonal Antibody Against The Dengue Virus

BackgroundDengue virus is an insect-borne virus of the flavivirus family,mainly transmitted by aedes aegypti and aedes albopictus,which causes dengue fever,dengue haemorrhagic fever and dengue shock syndrome in humans.Dengue virus is currently recognized by the world health organization as a major public health problem in tropical and subtropical areas.Globally,about 390 million people are infected with dengue virus each year,with about20,000 deaths per 50 million to 100 million infections.Although vaccine research against dengue virus has been carried out for nearly 70 years,there is no safe and effective vaccine against dengue virus or related therapeutic drugs.France’s Sanofi Pasteur company development of Live attenuated 4-valent vaccine-Dengvaxia has received in 2015 the FDA approved,But because of the serious adverse reactions inⅢphase clinical trials in Philippines and was halted.Due to the slow progress in the research of DENV active immune vaccine,people focus on the passive immunotherapy of DENV and the related research of therapeutic antibodies.As a mature,specific and effective passive immune strategy,monoclonal antibody has gradually become a research hot spot.For this purpose,four serotypes of DENV were used to immunize mice,and the natural immune activity of each serotype of DENV protein was preserved to the maximum extent.Mouse spleen cells were fused with SP2/0 cells to screen and prepare cross-reactive monoclonal antibodies against four serotypes of DENV,we identified its specificity and evaluated its neutralizing ability.In order to provide material basis and relevant information for passive immunotherapy,vaccine development and clinical diagnosis of dengue virus.Methods1.We immunized BALB/c mouse with four serotypes of DENV,after 1 week of the last immunization enhancement,blood was taken from the tail vein to separate the serum,We used indirect ELISA to detect the serum antibody titer of immunized mouse,mice with strong antibody responses to all four serotypes of DENV were selected as splenic cell donor for subsequent preparation of hybrid tumor cells.2.Spleen cell donor mice serum was isolated as a positive control,after the sterile separation of the spleen,spleen cells were fused with sp2/0 cells in logarithmic growth phase at a ratio of 1:5 under the action of PEG2000 to form hybrid tumor cells.3.The hybrid tumor cells after fusion were screened for positive subclones by limited dilution method,the antibody titer of the cell culture supernatant in each plate hole was detected by indirect ELISA,When the positive rate of cell culture supernatant in all the plate holes was 100%,the cell lines could be determined.4.The ascites antibody of BALB/c mice was generated by ascites induction,and purified by protein A affinity chromatography.SDS-PAGE electrophoresis was used to compare the ascites antibodies before and after purification,BCA method was used to detect the protein concentration of the purified antibodies.5.The Ig subclass of purified monoclonal antibody was identified by the mouse monoclonal antibody Ig/subclass identification kit.6.The titers of purified monoclonal antibodies against four serotypes of DENV were determined by indirect ELISA.7.The relative affinity constants of purified monoclonal antibody against four serotypes of DENV were determined by thiocyanate elution.8.The specificity of purified monoclonal antibodies was evaluated by indirect IFA and western blot.9.The neutralization ability of purified monoclonal antibodies was evaluated by PRNT.Results1.Serum antibody titers of immunized mice were significantly higher than those of unimmunized mice.The average antibody titer of 10 immunized mice against 4 serotypes of DENV was about 1:250,000.According to the experimental results,no.141 mice was selected as the donor of splenic cells for subsequent cell fusion.2.The fused cells were laid on a 96-well plate,and the cell fusion rate was 56%,the positive rate was 27%.After screening by positive subclones,3 DENV cross-reactive mabs were successfully obtained,which were named as 1G2,2F1 and 3G9,respectively.According to the titer test results of the supernatant antibody of cell culture,the cell line of 1G2 hybrid tumor was selected for the subsequent generation,purification and specific identification of ascites antibodies.3.Ascites antibodies were purified by Protein A affinity chromatography,SDS-PAGE electrophoresis gel showed significant antibody Protein heavy chain（55kD）and light chain（25kD）bands.The protein concentration of purified mAb was 3.0mg/ml by BCA method.4.The Ig subclass of mAb 1G2 was IgG_2b.the effective titers of purified mAb against DENV1～DENV4 were 1:640,1:640,1:320 and 1:32 respectively.The relative affinity constants of purified mAb against DENV1～DENV4 were 3mol/L,3mol/L,3mol/L and2.5mol/L,respectively.5.Indirect immunofluorescence staining showed significant cytoplasmic green fluores-cence in four denv-infected vero cells,and there was no green fluorescence in the uninfected vero cells,it indicates the mAb 1G2 is specific to DENV,not to the protein components of vero cells.However,no significant green fluorescence was observed in vero cells infected by YFV,WNV,HCV and ZIKV,indicating no cross-reactivity between mAb 1G2 and other members of the flavivirus family.6.WB results showed that there were significant target bands at about 55-60kD between mAb 1G2 and vero cell lysates of four serotypes of DENV infection after 48h。Based on the molecular weight comparison of each DENV constituent protein,we speculated that the target protein identified by mAb 1G2 should be E protein of DENV.Furthermore,we used four serotypes of DENV recombinant E proteins expressed by prokaryotes and mAb 1G2 for WB experiment,indicating that the target protein identified by mAb 1G2 was indeed the E protein of DNEV.In the next step,WB experiments were conducted with mAb 1G2 and DENV1 in Chongqing in 2019,YFV,WNV,HCV and ZIKV,indicating that the mAb 1G2 could identify DENV of other strains and had no cross-reactivity with YFV,WNV,HCV and ZIKV.7.The results of PRNT showed that mAb 1G2 had certain neutralization activity against four serotypes of DENV in vitro,and the PRNT₅₀ of DENV1 to DENV4 was 1:80,1:80,1:80and 1:40,respectively.ConclusionSuccessfully screened and identified a strain of mAb 1G2 that could cross-identify four serotypes of DENV,and its Ig subclass was IgG_2b.The target protein identified by mAb 1G2was DENV E protein,mAb 1G2 has good specificity and has no cross-reactivity with flavivirus family members YFV,WNV,HCV and ZIKV.The effective titers of mAb 1G2against DENV1 to DENV4 were 1:640,1:640,1:320 and 1:32,respectively.MAb 1G2showed a certain neutralization activity in vitro,and the PRNT₅₀ of DENV1 to DENV4 was1:80,1:80,1:80 and 1:40,respectively.