Screening And Preparation Of Neutralizing Antibodies Against West Nile Virus

West Nile Virus（WNV）is a positive single-stranded RNA virus belonging to the family Flaviviridae and genus Flavivirirus.It is transmitted by mosquitoes and can cause fatal neuroencephalitis in humans and horses,as well as death in birds and chickens.In recent years,WNV has been prevalent in temperate regions of Europe and North America,posing a great threat to human and animal health.However,there are no specific therapeutic drugs and commercial vaccines against WNV on the market at present.It is well known that neutralizing antibodies can prevent and treat WNV infection,and the preparation of WNV neutralizing monoclonal antibodies can be an effective means of WNV prevention and treatment.In this study,antigen preparation was performed first,WNV was inactivated withβ-propiolactone,and then concentrated by ultracentrifugation.Subsequently,6-week-old BALB/c mice were immunized with the prepared antigen,and the serum neutralizing titers of the mice were monitored in real time after immunization.The mice with higher neutralizing titers were selected to screen WNV neutralizing antibodies based on hybridoma technology and prepared in vitro.This study provides a new method for the prevention and treatment of WNV,provides ideas for the development of new vaccines,and provides a basis for further research on the invasion mechanism of WNV.The specific research content is as follows:1.Screening and preparation of WNV neutralizing antibodiesAfter WNV was inactivated byβ-propiolactone,the inactivated virus solution was concentrated by ultracentrifugation.6-week-old female BALB/c mice were immunized with the inactivated virus as antigen,and the neutralization titer of mouse serum was detected in real time.After four booster immunization,the neutralization titer of the mice serum tended to be stable and the neutralization titer of 3 mice serum reached 1:3200.Spleen cells from mice with high neutralizing titer were fused with myeloma cell line SP2/0,and positive hybridoma cells were screened for three subclones.The PRNT₅₀ of the hybridoma cells was measured by plaque reduction neutralization assay.Among them,8cell lines with high neutralizing activity were selected for mass preparation of monoclonal antibodies,and named as B2-D1-H6,C9-G11-F3,C11-C8-A3,C9-G11-D7,C11-C11-A9,C11-E8-B11,48-A11-E11 and 48-F12-E10.After neutralization titer assay and cross-protection assay with other flaviviruses,all of the eight monoclonal antibodies were able to neutralize WNV.Among them,B2-D1-H6 had the highest neutralization titer of 1:50-1:100.C9-G11-F3 showed good cross-protection against Japanese Encephalitis Virus（JEV）with neutralization titers of 1:50 against WNV and 1:40 against JEV.The neutralizing titer of C9-G11-D7 was 1:40.The neutralization titers of C11-C8-A3,48-A11-E12 and 48-F12-E10 were 1:20.The neutralization titers of C11-E8-B11 and C11-C11-A9 were 1:10.2.Preliminary resolution of epitopes of monoclonal antibodiesThe B cell epitopes and amino acid hydrophilicity of WNV E protein were analyzed by protein online prediction software.According to the results,E protein was divided into3 segments,named ED1,ED2 and ED3.Prokaryotic expression plasmids p ET-30a-WNV-ED1,p ET-30a-WNV-ED2 and p ET-30a-WNV-ED3 were constructed.After IPTG induction and inclusion body purification,WNV ED1,ED2 and ED3 proteins were obtained.B2-D1-H6 was confirmed to target ED1 by Western-Blot.Based on the prediction of bioinformatics software,we selected several possible sites of action in ED1 and synthesized short peptides,which were tested by indirect Enzyme Linked Immunosorbent Assay（ELISA）further verified that monoclonal antibody B2-D1-H6 acted on a 20-amino acid peptide E2 with the sequence of VCRQGVVDRGWGNGCGLFGK;In contrast,C9-G11-F3 does not recognize linear epitopes,but spatial epitopes.3.Evaluation of the therapeutic effect of monoclonal antibodies on WNV infection in miceThe therapeutic effects of monoclonal antibodies B2-D1-H6 and C9-G11-F3 against WNV infection in mice were verified by animal challenge test.In the mouse model,monoclonal antibody B2-D1-H6 and C9-G11-F3 had 20%protective effect against WNV infection and 40%protective effect against WNV infection,and C9-G11-F3 also had 20%protective effect against JEV infection.When the two antibodies were used together,the mice were protected against WNV infection with 66.7%protective effect.At the same time,the viral load and the expression levels of related inflammatory factors in the brain tissue of the treatment group were also significantly decreased.The results of hematoxylin-eosin（HE）staining and Immunohistochemistry（IHC）staining also showed:Compared with the negative control group,the antibody treatment group had milder pathological changes in brain tissue,and the activation and proliferation of microglia and astrocytes,which are markers of inflammatory activation,were also significantly reduced.These results demonstrated that monoclonal antibodies B2-D1-H6 and C9-G11-F3 prepared by us could have therapeutic effect on WNV infection in mice.4.Humanization of monoclonal antibodiesFirst,the humanized antibody skeleton was modified:Human Peripheral blood mononuclear cells（PBMC）were isolated from blood.The RNA of PBMC was extracted and converted into c DNA.The c DNA was used as a template to amplify the constant region sequence of the humanized antibody to complete the construction of the humanized vector backbone p FUSEss-CHIg-h G1 and p FUSEss-CLIg-h K.At the same time,B2-D1-H6 with the highest neutralizing titer and C9-G11-F3 with JEV and WNV cross-protection ability were selected to amplify the VH and VL sequences and cloned into the humanized vector backbone to construct a humanized recombinant anti-plasmid,which was transfected into HEK-293T cells.The supernatant was collected to verify the neutralization effect and cross-protection ability of the recombinant antibody.The results showed that the recombinant humanized antibody had WNV neutralizing ability.