Development、characterization And Analysis The Mechanism Of The Genetically Engineered Neutralizing Anti-PA63 Antibodies In Anthrax Treatment

Anthrax is an acute infectious diseases caused by Bacillus anthracis,Human infection occurs mainly through three major routes:the skin,the gastro-intestinal tract and the respiratory tract,resulting from contact with sick animals or animal products.It has a highly pathogenic ability,its spores could survive in the harsh environment and easily transmission.Therefore,bacillus was used to make biological weapons for a long time.Anthrax is defined ascategory A infectious disease in China[1-3].In the early stage,there is no obvious specificity symptoms,so its diagnosis often comes out late.This leads to patients missed the optimal cure time to use penicillin.Because penicillin can only kill bacillus anthracis,however,it has no effect on toxins released from Bacillus.After Bacillus Anthraxcoming into the blood stream,it can release three proteins,protective antigen(PA),lethal factor(LF)and edema factor(EF).These three proteins are nontoxic,if they exist alone.Once the protective antigencombined with lethal factor,they formed lethal toxin(LeTx).And the protective antigen combined with edema,they formed edema toxin(EdTx)[6-8].Therefore,only when the protective antigen combined to cell surface receptors,then form a heptamer channel on the cell surface,it can mediate lethal factor or edema factor.Protective antigen plays animportant role in anthrax and it is also the main immune original antigen which can induce protective immunity in humans or animals.To study antibodies against protective antigen have become critical in the treatment of anthrax.After PA combined with the cell surface,the Furin protease cleavageda 20 kDa protein from PA.The remaining 63 kDa protein(PA63)still combined to the cell surface.Obviously,the core of protective antigen is PA63.In this study,based on target of PA63,neutralizing murine anti-PA63 monoclonal antibody was prepared using hybridoma technology by immune mice with PA63.Consequently,it was transformed to human/mouse chimeric antibody with gene engineering technology.At the same time,PA63 was used to screen Fab from phage antibody library.It was also transformed to IgG via genetic engineering technology.The neutralization of genetic engineering antibodies was evaluated both in vitro and in vivo.Furthermore,antibody protection mechanism and neutralization epitope were analyzed.This research includes the following four parts.1.Expression and purification of PA63,PA,and LFFirstly,PA63 expression plasmid in prokaryotic expression system was constructed.The nucleic acid gel and gene sequencing was to prove PA63 plasmid successful construct.Then plasmid was transfered into E.coli,resulted in a 63 kDa protein expressed in it.The protein was identified by Western Blot with commercial anti-PA polyclonal antibody(Pierce)and anti-HIS antibody.We conformed it as PA63.After that,the conditions of PA63 expression was optimized.The most important conditions are induced time and induced temperature.In the end we determined the induced condition was induced at 37℃ for 4 h.Secondly,PA and LF expression plasmid in exocrine expression system was constructed.The gene of PA and LF were extracted from anthrax vaccine strain.Moreover,we replaced the plasmid promoter to PA promoter.Then plasmid was transfected to weakened anthrax bacillus strain.Consequently,these two expressed proteins were detected by Western Blot with PA and LF commercial rabbit polyclonal antibody(Pierce),respectively.Furthermore,the lethal characterization of PA and LF were identified.PA and LF protein at the concentration of 0.01μg/ml could kill 50%J774A.1.And LF concentration reached to 10μg/ml,the cell survival rate was below 20%.In Fischer344 rat model,PA and LF at a concentration of 30μg/each rat could kill rats in about 90 min.With these experiments,we prepared PA63,PA and LF.Moreover we confirmed their fuction.2,Preparation,expression and identification of anti-PA63 neutralizing genetic engineering antibodies1)Preparation,expression and identification of anti-PA63 neutralizing murine/human chimeric antibodyIn this stage,we used PA63 to immune mice,and developed mouse monoclonal antibodies with hybridoma technology.There were four murine anti-PA63 monoclonal antibodies,named PA5,PA6,PA7,PA8,showed well neutralizing in vitro.Lethal toxin at a concentration of 10μg/ml,and the antibody concentration in 20μg/ml,PA5 and PA6 can protect more than 95%cells alive,PA7 and PA8 can protect more than 80%cells alive.Finally,PA6 was chose to transformed to chimeric antibody.The eukaryotic expression plasmids of chimeric antibody were constructed by gene recombination.More,the expression system was optimized.The ratio of expression plasmid and transfection reagents was 1μg:1μl.We harvest cell supernatant after six days and then purefied with Pro.A affinity colomn.The modified genetic engineering antibody,named hmPA6,with affinity of 1.438 x 10-10M could recognized PA63 as well.2)Preparation,expression and identification of anti-PA63 neutralizing full human IgG antibodyIn this stage,Fab antibodies were selected by six rounds of "adsorption elutionamplification" in phage antibody library.Three Fab,cloning,16,21 and 32,was obtained.In vitro study,lethal toxin at a concentration of 10μg/ml,Fab at a concentration of 10μg/ml,the cloning of 21 protected more than 90%cells alive,cloning 16 and 32 protected 80%cells alive.Therefore clone 21 was chose to transform to genetically engineered IgG antibody.The full human IgG,named PA21,could combine PA63.There was a dose effect relationship between PA21 and PA63.And the affinity of PA21 was 1.0003 x 10-9 M.In this section,it was confirmed PA63 can be used to prepared neutralizing monoclonal antibody with its immunogenicity.PA63 could also be used to screen Fab from phage antibody library.Even if we transformed antibodies to genetic antibody,they keep their binding and affinity.Furthermore,we could detect their protection fuction.3.Neutralizing effect of anti-PA63 engineering antibodyIn this stage,the neutralizing of hmPA6 and PA21 was tested in vitro primarily.The mice macrophage J774A.1 cell was used to detecte their protection.The results showed that the toxin at 10μg/ml,the antibody at 4 μg/ml,PA21 made more than 90%cell alive,hmPA6 protect 80%cells alive.And in vivo,at first,antibody and toxin injection at the same time,the results showed that the PA21 could protect all rats at 10μg/each rat,while hmPA6’s dose was 45μg/each rat.Then antibody injection before toxins,the experimental results showed that 90μg/each rat of hmPA6 injected 48 h hour before lethal toxin could protect all rats.20μg/each rat of PA21 injected 24 h before before toxins could protect all rats.After that,antibody was injected after toxin.The results showed that PA21,at 20μg/each,could still be able to protect rats.Finally the metabolism in rats serum of hmPA6 and PA21 was detectd.Based on ELISA standard curve,we calculated antibody concentration in serum.The results showed that the serum half-life of hmPA6 was 48 h,while PA21 was 24 h.The above experiments show that genetic engineering antibody hmPA6 and PA21 had good neutralizing function.This was the key to study the protection mechanism.4.The protection mechanism and neutralizing epitope of anti-PA63 gene engineering antibodyIn this stage,we mainly studied the protection mechanism and neutralization epitope of hmPA6 and PA21.First by competitive ELISA and immune coprecipitation experiments,it was confirmed that hmPA6 and PA21 had no competitive relationship with LF when binding with PA63.With the increased LF,the combined antibodies to PA63 were almost the same.Then Furin protease cracking PA experiments show that,with the increased amount of antibodies,the cleavaged PA were almost the same.Therefore,hmPA6 and PA21 cannot suppress Furin protease.Then using Western Blot to dectectPA which combined to cell surface.With the increase of hmPA6 antibody,the detected PA and PA63 on cell surface were significantly reduced.When hmPA6 increased to a certain amount,the PA and PA63 were almost unable to detect.However,the PA21 was without this phenomenon In all,we confirmed hmPA6 could inhibit PA combinding to cell surface,and we indirectly proved that PA21 could interfere with PA63 oligomerization.We further analysed the two antibodies’neutralizing epitope.Constructed respectively the different domains of PA,Western Blot and ELISA experiment confirmed that hmPA6 could recognize domain Ⅳ of PA;PA21 recognized domain Ⅱ and/or domain Ⅲ.In summary,we first developed anti-PA63 neutralizing monoclonal antibody by hybridoma technology,then transformed to human/mouse chimeric antibody,named hmPA6.Second,we screened Fab phage antibody library and obtained anti-PA63 Fab.Consequently,we transformed it to full human IgG,named PA21.These two engineering antibody,hmPA6 and PA21,had good neutralizing fuction in vitro and in vivo.Further,we confirmed that hmPA6 recognized domain IVof PA and then prevent PA from combining with cell surface.However,PA21 recognized domain Ⅱand/or domain Ⅲ of PA,then interfered with the oligomerization of PA63.These experiments provide solid foundation for anthrax antibody drug development.