Screening Of Human Monoclonal Antibodies Against Staphylococcus Aureus Based On A Single Peripheral Plasma Cell Technique

Staphylococcus aureus(S.aureus)is a pathogen that is widespread and seriously endangering human health,especially methicillin-resistant Staphylococcus aureus(MRSA).It is a "superbug" with strong pathogenicity and multi-drug resistance,which has become a difficult problem in clinical treatment and seriously threatens human health.In 2017,the World Health Organization(WHO)listed “the 12 most dangerous 'superbugs' ”,and Staphylococcus aureus was listed as “high” priority.About 94,000 people in the United States are seriously infected with MRSA each year,resulting in a death toll of about 19,000.The incidence of MRSA infection in China is relatively high.The survey of “National Bacterial Resistance Detection Network” shows that the clinical isolation rate of Staphylococcus aureus in Gram-positive bacteria in mainland hospitals is the highest,and the incidence of MRSA infection in hospital is about 8%.The direct economic loss caused by the infection is as high as 15 billion yuan.In the face of increasing drug resistance of Staphylococcus aureus,research on immune control strategies needs to be strengthened to develop new,effective and resistant drug-resistant immunosuppressive drugs,such as therapeutic antibodies,for effective control of Staphylococcus aureus infection.It is of great significance to reduce the mortality rate of hospital infections and delay the development of drug resistance.At present,no anti-S.aureus drugs have been used in clinic.In the clinical study of therapeutic antibodies to MRSA carried out abroad,INH-A21 of Inhibitex Company targets surface agglutination factor A(ClfA).Phase III clinical trials showed that there was no significant difference between the experimental group and placebo group.Human immunoglobulin targeting S.aureus capsule of Nabi Company also declared defeat after Phase II clinical trial.At present,the clinical trials of MRSA vaccine,SA4 Ag of Pfizer Company and GSK2392103 A of GSK Company,contain four key antigen components of S.aureus.There are many virulence factors and complex pathogenic factors in S.aureus,so it is difficult to form effective protection for single antigen target.Therefore,based on the results of a large number of preclinical and clinical trials on immune prevention and treatment of S.aureus,and summarizing the failure reasons of relevant studies,the therapeutic antibody drugs of S.aureus should be able to cover several key virulence factors of S.aureus strains in order to form effective protection.In our previous studies on immune prevention and treatment of S.aureus,five immunodominant antigens with good immunogenicity and high conservativeness were screened from 2742 open reading frames of the whole genome of S.aureus by using reverse vaccination technology and a large number of animal immune protection experiments: alpha-hemolysin(Hla),iron surface determinant protein B(IsdB),S.aureus protein A(SpA),enterotoxin B(SEB)and manganese ion-binding protein C(MntC).Single antigen immunized mice showed certain protective effects on S.aureus.The recombinant S.aureus vaccine was developed with the antigens of the corresponding detoxification mutation as the main component,which has good immune protection in animal S.aureus bacteremia,pneumonia infection and fracture infection models.Phase I clinical trial o f human body was successfully accomplished.Objectives:In this study,the peripheral blood of subjects in phase I clinical trial of recombinant MRSA vaccine was used as the source of antibody.The antibody expression vector was constructed by single plasma cell technique.The expression vector was transfected into 293 T cells instantaneously.The supernatant of cell culture was screened by ELISA.After a large number of positive antibodies were expressed,the binding activity and neutralization activity were evaluated in vitro.And the mice alpha hemolysin lethal model and MRSA252 bacteremia lethal model were used to evaluate the immune protective effect of monoclonal antibody in vivo.It lays a solid experimental foundation for the development of S.aureus therapeutic monoclonal antibody drugs.Methods:1.Five mutant protective antigens mHla,IsdB,SpA5,mSEB and MntC of S.aureus were coated,and the antibody titer in peripheral blood serum of Phase ? clinical trial was detected by ELISA.Peripheral blood mononuclear cells(PBMC)with high serum antibody titer were screened for subsequent antibody development.The antibodies of plasma cell surface markers such as CD19,CD27 and CD38 were labeled with fluorescence.The labeled antibodies were co-incubated with PBMC samples corresponding to the high titer serum of antibody.The lymphocytes in PBMC were fluorescent.Flow cytometry was used to detect CD19+,CD27+ and CD38+ in PBMC,and CD3,CD14 and other distinguishing markers negative cell groups were sorted into single plasma cell.Using genetic engineering technology,RT PCR was carried out to amplify antibody variable region gene cDNA using the mRNA in single plasma cells as template.Then,cDNA was used as template to amplify the antibody variable region gene by specific primers after two rounds of PCR.The DNA was electrophoretic,and the samples with positive heavy chain and light chain were sequenced.Using bioinformatics method,sequencing results were compared with the sequences in the antibody gene library,and the information of antibody gene subtypes was analyzed.The variable region gene of antibody was linked with promoter,constant region gene and poly(A)tail by overlapping PCR to construct a complete linear antibody expression vector.The expression vector was transfected into 293 T cells,and five protective antigens of MRSA were coated.The supernatants of 293 T cells were screened by ELISA to obtain positive monoclonal antibodies.The linear expression vector of positive monoclonal antibody was cloned into pcDNA3.3 plasmid by genetic engineering technology and expressed by 293 T cells.3.By encapsulating five antigen proteins of S.aureus,ELISA was used to detect the absorbance value of monoclonal antibodies at different concentrations after binding with corresponding antigens to evaluate the binding activity of monoclonal antibodies to antigens.wtHla and it's monoclonal antibodies were co-incubated before applied to rabbit red blood cells and A549 cells to detect the blocking effect of monoclonal antibodies on the hemolysis of wtHla and A549 cells,by which evaluates their neutralization activity.4.Establish the lethal model of mice exposed to wtHla intraperitoneally and intravenously.After co-incubation of Hla monoclonal antibody with wtHla in vitro,the mix was challenged to mice,and the immune protective effect of Hla monoclonal antibody on mice exposed to wtHla was evaluated.The lethal model of MRSA252 mice with bacteremia infection was established.Antibody was injected intravenously 2 hours after or 24 hours before the lethal dose of MRSA252 challenge.The immune protective effect of the antibody on mice with MRSA252 bacteremia infection was evaluated.Results:1.Fifty-two serum samples of peripheral blood from volunteers immunized with recombinant S.aureus vaccine in phase I subject were screened by ELISA.Eight serum samples with high antibody titer against five protective antigens were obtained.After thrice immunizations,the titer of serum specific antibodies was 16-256 times higher than that before immunization.Eight PBMCs corresponding to high titer antibody serum were labeled by fluorescence and sorted by flow cytometry.A total of 1120 plasma cells were selected.2.RT-PCR and two rounds of PCR were performed using the RNA of plasma cells as template.Then 461 antibody heavy chain and light chain variable region genes were obtained by gene sequencing and antibody sequence alignment.Heavy chain and light chain variable region genes were assembled into linear expression vectors by overlapping PCR and transfected to expression.39 positive monoclonal antibodies were screened by ELISA,and antibody subtypes and heavy chain genes were analyzed.The analysis of antibody subtypes showed that there were 15 IgG1,3 IgG2,14 IgA and 7 IgM,and 5 VH1,3 VH2,23 VH3,9 VH4 and 1 VH5 subtypes in the heavy chain variable region of antibodies,and 11 VK1,4 VK2,3 VK3,1 VK4,8 VL1,6 VL2,4 VL3,1 VL9 and 1 VL10 subtypes in the light chain variable region of antibodies.10 of these monoclonal antibodies were expressed more than 1 mgs.3.The binding activity of 10 monoclonal antibodies detected by ELISA showed that Hla monoclonal antibody M0411,IsdB monoclonal antibody M0318,SpA5 monoclonal antibodies M0659,M0662,SEB monoclonal antibody M0283 and MntC monoclonal antibody M0686 had better binding activity.When the concentration of antibody increased,the absorbance value at 450 nm also increased.Western-blot results showed that all 9 monoclonal antibodies recognized linear epitopes except the conformational epitope of SpA5 monoclonal antibody M0662.The blockade of wtHla hemolysis and A549 cells killing activity showed that M0411 had a good neutralization effect and could neutralize the toxicity of wtHla in vitro.4.The lethal models of tail vein and abdominal cavity of wtHla were established with the dosage of 4 ?g/rat and 14 ?g/rat respectively(mortality?90%).In the model of intraperitoneal challenge death,20?g/rat M0411 had 100% protective effect;in the model of tail vein attack death,10?g/rat M0411 and 5?g/rat M0411 had 100% protective effect.A lethal infection model of bacteremia was established with MRSA252 of 9x108 CFU per mouse(mortality rate?90%).In this model,20 mg/kg body weight of M0411 was injected 24 hours before the challenge,the survival rate of which reached 100%,and that of mice injected with 10 mg/kg body weight of M0411 was 90%.There was no significant difference in survival rate of mice injected with 10 mg/kg body weight M0411 2 hours after challenge comparing with control group.Conclusions:1.Eight serum samples with high antibody titer were screened by ELISA,and 1120 plasma cells were selected from corresponding PBMC.2.Through PCR amplification,gene sequencing and sequence alignment,461 antibody heavy chain and light chain variable region genes were obtained,and linear expression vectors were constructed.39 monoclonal antibodies were screened from the supernatant of cells transfected with the expression vectors,and 10 of them were expressed and purified in large quantities,with high purity.4.The binding activity of M0283,M0318,M0411,M0659,M0662 and M0686 was better.Western-blot results showed that M0662 recognized conformational epitopes and the other 9 monoclonal antibodies recognized linear epitopes.5.Neutralization experiments in vitro showed that M0411 could block the hemolysis activity of wtHla on rabbit red blood cells and the killing activity towards A549 cells,and had good neutralization activity.6.The results of in vivo immune protection experiment in mice showed that M0411 could block the lethal effect of wtHla on mice.In the meanwhile,the immune protection rate of M0411 on MRSA252 challenge mice reached 100%.