Construction Of Fully Human Genetically Engineered Antibodies Against Pneumolysin O And Analysis Of Their Biological Activitie

Streptococcus pneumoniae can cause otitis media,pneumonia,meningitis,endocarditis,and sepsis.According to WHO statistics,about 1 million infants worldwide die from S.pneumoniae infection every year,of which about 30,000infants die in China.In recent years,the drug resistance of S.pneumoniae has been increasing seriously,and the pathogen causing pneumonia has become complex and difficult to accurately diagnose quickly,which could lead to improper or delayed medication and cause lethal S.pneumoniae infection.Pneumolysin O（PLY）has proved to be an important virulence in the pathogenesis of S.pneumoniae:it can destroy the integrity of the tracheal epithelium and blood-brain barrier facilitating bacteria enter into blood stream and invasion of the brain;activate NLRP3inflammasome resulting in overproduction of proinflammatory cytokines,kown as cytokine storm.PLY toxin was choosed to conjugate with traditional S.pneumoniae vaccines to develope novel protein conjugate vaccine.The results of animal experiments,phase 1 and phase 2 clinical trials indicated that the new vaccine has better immunogenicity and safety.It is expected that fully human neutralizing antibody against PLY would help to increase the treatment efficiency of the S.pneumoniae infection before the novel protein conjugate vaccine was authorized in clinical practice.The fully human monoclonal antibody against SLY obtained earlier can effectively block the hemolytic activity of r PLY.In this study,the variable region sequence of these antibodies were used to construct genetically engineered antibodies and verify its neutralization against r PLY by cell test and animal test.The main research methods and results are as follows:1.Preparation of r PLY and its functional domainsThe ply gene of the S.pneumococcal D39 and the gene fragment encoding PLY D1-3 and PLY D4 were cloned between the Xho I-Bam H I sites of the p ET28a plasmid,the recombinant plasmids p ET28a-ply,p ET28a-ply d1-3,and p ET28a-ply d4were successfully constructed.The recombinant plasmid was introduced into E.coli BL21（DE3）and the recombinant bacteria were induced at 16℃for 18 h.The bacterial were broken after sonication and found that the three recombinant proteins r PLY,r PLY D1-3,r PLY D4 were solubly expressed.The expression products were purified by Ni²⁺-NTA affinity column.SDS-PAGE and hemolysis tests showed that the molecular weight of each recombinant protein matched the theoretical value,with purity above 90%and r PLY having hemolytic activity.2.Development of fully human genetically engineered antibodies against PLYThrough western blotting experiments,enzyme-linked immunosorbent methods and hemolytic blocking assays,we found that the three fully human monoclonal antibodies against SLY obtained in the laboratory have high affinity for r PLY and have ability to block its hemolysis.The light chain and heavy chain variable region genes of antibodies were cloned into the modified p CDNA3.4 vector,acquired six recombinant plasmids.HEK293F cells were co-transfected with expression vectors of three antibodies’light and heavy chains,respectively.After 7 days,cell culture medium was collected and purified by Protein A affinity chromatography column to obtain genetically engineered antibodies 1-10F,2-2G and 4-2G.Antibody molecular weight was determined by SDS-PAGE with purity above 98%.The EC₅₀values of r PLY for 1-10F,2-2G and 4-2G were detected by ELISA were 41.61μM,15.52μM and 48.40μM,and Ka values were 1.99×10⁵,3.99×10⁵and9.31×10⁴.2-2G has the highest affinity,followed by 1-10F and 4-2G.Western blotting revealed that the epitopes recognized by these three antibodies are present in the 1-3domain of r PLY and they do not bind the fourth domain.3.In vivo and in vitro neutralization of genetically engineered antibodiesNeutralization of three fully human genetically engineered antibodies in vitro was determined by red blood cells and HEK293 cells,which showed that the blocking rate of 500μg/m L 1-10F on 2.7 n M r PLY（equivalent to 1 hemolytic unit）reached more than 95%and 10.8 n M r PLY over 80%.The other two strains had slightly worse blocking effect,with 250μg/m L 2-2G blocking only 45%for 2.7 n M r PLY and 20%for 10.8 n M r PLY,and the blocking rate did not be significantly improved after the antibody dose increased.The blocking effect of antibody 4-2G was bounded between the two,reaching over 75%for 2.7 n M r PLY at 500μg/m L and 60%for 10.8 n M r PLY.For HEK293 cells,750μg/m L 1-10F blocked 70%of 1090 n M r PLY(equivalent to 2 IC₅₀),and the other two antibodies had no significant blocking effect.Neutralization in vivo was performed using a prophylactic passive immunization assay to assess the protective effects of antibodies against inflammatory responses and tissue damage caused by r PLY.Five-week-old BALB/c mice were subcutaneously injected with 160μg 1-10F,control mice were injected with normal saline,45μg r PLY were intraperitoneally injected after 24 h.IL-1β,TNF-α,IFN-γand IL-8concentrations in serum were measured at 1,3 and 5 h after challenge,and heart,lungs,liver,spleen were taken at the last time point for immunohistochemistry and pathological analysis after paraformaldehyde fixation.The results showed that the serum levels of IL-1β,TNF-ɑ,IFN-γand IL-8 in control mice gradually increased over time after challenge,reaching 497.4 pg/m L,1836.3 pg/m L,609.1 pg/m L and 168.2 pg/m L respectively in 5 h.On the contrary,the levels of cytokines increased slightly in the antibody group,and the concentrations of the four cytokines in serum samples were 153.8 pg/m L,1193.8 pg/m L,216.7 pg/m L,and 83 pg/m L respectively in 5 h,all were significantly lower than the challenge control group.Immunohistochemistry analysis showed no significant positive rate in heart and lung,histopathological analysis showed no significant pathological changes in heart,lung,liver and spleen in all groups.These results show that in a short period of time,r PLY is mainly toxic by stimulating the inflammatory response rather than causing histopathological damage,and antibodies play a passive immunoprotective effect by reducing the concentration of inflammatory factors by neutralizing r PLY.In conclusion,three fully human genetically engineered antibodies against r PLY were successfully developed,of which 1-10F have the greatest neutralizing property in vitro and in vivo.It has the potentiality of developing drugs as an adjuvant therapy for pneumococcal disease.