Neutralizing Epitopes Of Anthrax Toxin Protective Antigen

Anthrax toxin consists of three proteins,protective antigen(PA),lethal factor(LF),and edema factor(EF).PA is the major component of the current anthrax vaccine,but the antigenic epitopes on it are not well defined.We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies(mAbs)to analyze the neutralizing epitopes of PA.In this work,anti-PA mAbs were prepared according to the standard hybridoma technique, and nine toxin-neutralizing mAbs were obtained.We then determined the domains recognized by those mAbs with an ELISA detection,which we used a series of recombinant proteins comprising different domains of PA as coated protein and the results was confirmed by western blot.It was found that four mAbs(5E12,2A8,5E1,1B3)bound to domain 2 of PA,one(4D10) bound to domain 3,and the other four(4B2,4B6,4F12,3E3)bound to domain 4.Subsequently, we investigated the relationship between these mAbs by competitive binding assay,and found five potential neutralizing epitopes.The purified mAbs were tested their neutralizing activity against lethal toxin by toxin neutralization assay,and used rabbit anti-PA polyclonal antibody as a control.Three mAbs(5E12,2A8,5E1)recognized the same epitope within domain 2 were observed a higher TNA titer than that of polyclonal antibodies and the other mAbs.The results support the proposal that PA may have several neutralizing epitopes,and suggest that there are dominant neutralizing epitopes in domain 2 of PA.Firstly,we used Phage display technique to screen toxin-neutralizing mAbs(5E12,2A8 and 5E1)which bind to the same epitope in PA domain2 with a higher neutralizing activity.As a result,we got a four amino acids consensus sequence"SFFD" which is identical with the 312-315 amino acids residues sequence in PA.Besides,it is known that this sequence located in PA domain2 2β₂-2β₃ loop,and relate to the cleavage site of chymotrypsin,we used proteolytic assay,proteolytic protection assay and constructed SFFD deleted PA mutants to verify whether the "SFFD" was the critical amino acids sequence for mAbs 5E12,2A8,5E1's, recognizing.The result was consisted with our expectation.In the phage immunization,a phage clone containing such sequence was amplified and was used as immunogen to inoculate mice, the results showed that mice immunized with specific phages generated anti-PA antibodies. The result demonstrated for the first time that the 2β₂-2β₃ loop,which is involved in the transition of PA oligomers from prepore to pore,contains a dominant neutralizing epitope.In order to locate the sequence "SFFD" in PA,we use a software Accelrys Discovery Stdio to analyze the structure of PA,and through an analysis of a series of amino acids in PA monomer which 7 A away from the "SFFD",we found that the amino acids 410,414,496,498,633, 637,668,670,672 and 673 from the neighbouring monomer may all have interaction with this sequence.This result also implied that the amino acids 312-315 from the 2β₂-2β₃ loop may partially or fully take an effect in toxication.Furthermore,5E1 could block the formation of SDS-resistant heptamer,it is also show that this epitope may play an important role in the transition of PA oligomers from prepore to pore.PA could induce protective immunity to the anthrax infection and is the major component of current anthrax vaccine(anthrax vaccine adsorbed,AVA).Here,rPA was used as an immunogen to inoculate mice and guinea pigs,data from both animal models show that the titer of rPA specific antibodies began to increase after the first inoculation,and reached the maximal after boosting.On the other hand,the titer of epitope-specific antibodies started to increase since the second inoculation and reached a higher level four weeks after the first injection.This trend was similar to that of neutralizing antibodies.Besides,we use the same method to screen the 4D10 specific epitope in PA,and we got a sequence"NETNI" which is similar with the 570-574 amino acids in PA domain3.Aim to verify such results,we constructed a series of PA truncated mutants to test their capability of binding with mAb 4D10,respectively.And the result is consistent with our expectation.In this research,we also found a synergism between 4D10 and other mAbs on toxin neutratrlization.In PA domain 4 neutralizing epitopes' screening,mAb 4B2 was used.Through three rounds of panning,a consensus sequence "PLYISN#N" was found,which is similar with the sequence 686PLYISNPN693 in PA domain4.For further research,we also constructed a series of truncated or deleted PA mutants,and we found only PAdelSNPN could recognize 4F12. This result showed that the C-terminal of PA maintained the structure of whole PA molecule and the sequence "PLYISNPN" is the key residues recognized by both 4B2 and 4F12.It is known that 682N and 683D played a vital role in PA binding to ATR,so research pointed to the PA mutants PAD683A,PAD683N,PAN682D and PAN683A,indicated that the 682N is an important amino acid in 4B2 or 4F12 contact with its epitopes.Moreover these two MAbs could also block PA binding to its receptor,and it suggested that there was a dominat epitope in 4β₈-4β₉ loop.Data from the crystal structure of PA show that domain4 has less opportunity to contact with other domain.It was known 4β₈-4β9 loop was related with the cell-binding activity,so we presume that this epitope may play an important role in the early phase of the toxication.