Features Of B Cell Immune Repertoire And Protective Mechanism Of Inactivated Rabies Virus

BackgroundRabies is an ancient zoonotic infectious disease with a case fatality rate of nearly 100%.It is prevalent in more than 150 countries around the world,and about 59 000 people die from rabies every year.Rabies is mainly caused by Rabies virus(RABV)infection,which is transmitted by bites,scratches of infected animals or contact of infectious saliva with broken skin and mucous membranes.95%of human rabies is transmitted by dogs,and dog borne rabies has been effectively controlled in recent years.However,the increasing number of cases transmitted by wild animals such as bats,foxes and raccoons,as well as the risk of spillovers from other hosts,complicate the prevention and control of rabies.Despite the high fatality rate,rabies can be effectively prevented through pre-exposure and post-exposure vaccination.According to statistics,about 19-50 million people receive rabies post-exposure prophylaxis(PEP)every year,and the cost is as high as 8.6 billion US dollars,resulting in a heavy social burden.At present,the inactivated rabies vaccine without adjuvant is used for human use at home and abroad,but the inactivated vaccine has the defects of weak immunogenicity,rapid decline of neutralizing antibody and short duration of protection.The immunity of inactivated rabies vaccine is still improved by the way of ’primary immune-boost’.The process of immune response to this multiple-dose inactivated rabies vaccine has not been precisely characterized.The main function of B cells is to mediate the humoral immune response to induce the production of high-affinity antibodies.During the development and differentiation of B cells,B cells can form a diverse B cell surface receptor(BCR)repertoire.Therefore,it is urgent to explore the relationship between the B cell immune response and the generation,maturation and maintenance of antibodies after immunization with inactivated rabies vaccine,as well as the dynamic characteristics and immune protection effect,so as to provide a basis for the design of highly effective rabies vaccines and the development of passive immune preparations,and reduce social costs.Objectives1.High titer inactivated rabies virus was prepared based on RABV LBNSE vaccine strain,and the antibody level,the changes of B lymphocyte subsets and the protective effect in vivo were studied by using BALB/c mice as an animal model.2.It is to analyze the clonotype,diversity,complementarity determining region,gene usage and combination characteristics of inactivated rabies virus vaccine without adjuvant by immune repertoire sequencing.Combined with the changes of antibody and cell subsets,the characteristics of BCR repertoire were explored.Methods1.Preparation of inactivated rabies virus vaccine:high-titer rabies virus vaccine strain LBNSE was cultured in vitro with sensitive cell NA,the titer of the virus was determined,and the virus was inactivated by β-propiolactone,then blind passaged for three generations,and the inactivation effect was identified by immunofluorescence assay(IFA).2.Evaluation of infection protection ability of inactivated rabies virus vaccine:BALB/c mice aged 6-8 weeks were divided into inactivated virus group and control group.The mice were immunized intramuscularly at 0,7,21 days,and RABV challenge strains were injected intramuscularly at 4w after immunization.The mice were observed continuously for 21 days after infection,and the changes in body weight,clinical symptoms and death were recorded.And RABV N gene was identified by RTPCR in the brain tissue of mice to evaluate the protective effect of inactivated rabies virus on mice.3.The changes of antibody level induced by inactivated rabies virus vaccine:6-8 weeks old BALB/c mice were used as the animal model,and the peripheral blood was collected at 0w,1w,2w and 4w after immunization and serum was separated.The neutralizing antibody titers were determined by fluorescent antibody virus neutralization test(FAVN),and the binding antibody titers were determined by enzymelinked immunosorbent assay(ELISA)to evaluate the characteristics of the induced antibody levels during immunization.4.Changes of antibody subtypes induced by inactivated rabies virus vaccine:ELISA was used to detect the changes in the titer of IgG1,IgG2a,IgG2b,IgG3 and IgM antibody subtypes and the changes in the proportion of IgG1 and IgG2a.5.Changes in B lymphocyte subsets induced by inactivated rabies virus vaccine:Splenic lymphocytes were isolated at 0w,1w,2w and 4w after immunization.Flow cytometry was used to detect the changes in the proportion of total B cells,follicular zone B cells,marginal zone B cells and plasma cells during immunization.6.Changes in IgG memory B cells induced by inactivated rabies virus vaccine:Splenic lymphocytes were isolated at 0w,1w,2w and 4w after immunization.The number of total IgG memory B cells and RABV G protein-specific memory B cells were determined by enzyme-linked immunospot(ELISpot)assay.7.Changes in BCR repertoire after immunization with inactivated rabies virus vaccine:Peripheral blood mononuclear cells(PBMC)were isolated from mice at 0,1,2 and 4 weeks after immunization.Nested PCR was used to construct BCR library,and Illumina platform was used to sequence the immune library.After filtering and quality control,the data were visualized by Loupe VDJ Browser software and analyzed by scRepertoire package.Results1.High titer LBNSE virus was cultured successfully,and the titer of LBNSE strain was 109.5TCID50/ml.After inactivation,the virus was blind propagated on NA cells for three passages,and no green fluorescence was detected by IFA,indicating that the virus was inactivated successfully.2.The results of infection protection experiment showed that the inactivated LBNSE virus could protect mice from RABV challenge strain infection.During the observation period,the body weight of mice changed slightly,no clinical symptoms appeared,the survival rate of mice was 100%,and RABV N gene was not amplified by RT-PCR.3.The titers of neutralizing antibody and binding antibody showed a rising trend during immunization.The titers of antibodies induced at 1w after immunization were low,while those at 2w and 4w after immunization were significantly increased.The neutralizing antibody titers reached and exceeded the protection value of 0.5IU/ml specified by WHO at 2w.4.The results of antibody subtype detection showed that the IgM titer was the highest at 1 week after immunization,while the IgG1 and IgG2b titers were significantly increased at 2 and 4 weeks after immunization,and there was a Th2 type immune response tendency.5.The results of flow cytometry showed that the proportion of B cells was the highest at 4w after immunization,and the proportion of B cells in the follicular zone was significantly increased,and the proportion of B cells in the marginal zone was significantly decreased,and the proportion of differentiated plasma cells reached the peak at 2w after immunization,with an average of 1.39%.6.ELISpot assay showed that the number of total IgG memory B cells was significantly increased at 2 weeks after immunization,while the number of RABV Gspecific memory B cells increased significantly with the increase of immunization dose and reached the peak at 4 weeks after immunization.7.The analysis results of the sequencing data of the immune repertoire showed th at the number of clonotypes increased after immunization with inactivated rabies viru s,and it showed the distribution characteristics of mainly single clone type and less hi gh frequency clone type.With the increase of immunization doses,the length of CDR 3 region was continuously increased,and the diversity index continued to decrease,re sulting in clonal expansion.The overlap index increased and the number of shared clo nes increased at 1 w,2w and 4w after immunization.Comparison of the frequencies of heavy chain and light chain VDJC gene usage and combination revealed several key c hanged genes including IGHV1-5,IGHV5-6,IGHV3-1,IGKV9-120,IGKV1-117,IG HM,IGHD and multiple up-regulated V-J gene combinations such as IGHV7-3-IGHJ 4,IGHV2-2-IGHJ4 and IGKV1-135-IGKJ1 and dominant paired gene such as IGHV5-17/IGHJ3/IGHG2B/IGKV1-110/IGKJ2 and IGHV5-17/IGHJ3/IGHG2B/IGKV4-58/I GKJ4 after immunization.ConclusionIn this study,a high titer inactivated rabies virus vaccine without adjuvant was successfully prepared based on LBNSE strain.It was found that the length of BCR CDR3 region was continuously increased,the diversity was decreased,the clonal expansion was increased,and the preferred gene fragments and combinations were changed after immunization with inactivated rabies virus in mouse model.Humoral immune response is strongly activated,B cell differentiate into plasma cell to produce high levels of antibodies to provide effective protection,and is continuously recruited to participate in intrafollicular reactions and undergoes affinity maturation and class switching to induce the continuous differentiation of specific memory B cells to provide more durable immune protection.Through in-depth study of the immune response process of inactivated rabies vaccine,the basis and ideas for the design of more rapid and efficient rabies vaccines and the development of therapeutic rabies antibody drugs are provided.