Discovery Of Bacterial Surface Protective Antigens By Using A Combined Approach Of Phage Display And Subcellular Localization

Bacterial surface proteins can be used for developing good vaccine candidates.In the present study,we highlight research exploiting phage display technology combined with subcellular localization as a rapid,inexpensive and efficient means for discovering surface protective antigens of bacteria.To demonstrate the method,Erysipelothrix rhusiopthiae(E.rhusiopathiae)and Pasteurella multocida(P.multocida)serotype A were selected as model pathogens.Phage-displayed random dodecapeptide and loop-constrained heptapeptide libraries were immunoscreened with polyclonal antibodies against surface molecules of E.rhusiopathiae and P.multocida.We used a new mimotope screen and 96-well plate amplification approach to maintain the diversity of mimotopes: library phages were firstly mixed with antibodies in solution,and phage-antibody complexes were then captured by coated Staphylococcus Protein A(SPA)and Streptococcus Protein G(SPG).Following washing and eluting,the obtained phages were diluted 1000 fold and amplified in ten 96-well plates,resulting in a significant decrease of competitive amplification of each phage and even of non-competitive amplification.The obtained mimotopes were used for homology analysis and subcellular localization.A total of 14 newly defined surface proteins of E.rhusiopathiae and 7 newly defined surface proteins of P.multocida serotype A and D were selected for gene clone,express and immunogenic analysis.Nine new protective antigens of E.rhusiopathiae were identified(CwpA,Plp,CbpA,Bga,Bml,Neu,CwpB,Da and Atsp)from 11 proteins.Five new protective antigens of P.multocida were identified(LppC,TonB-d,pTonB,Omabp and Tp)from 6 proteins.In our approach,the screening efficiency of protective antigens of E.rhusiopathiae and P.multocida were >80%.Compared to the screening efficiency of reverse vaccinology and proteomics technologies(the screening efficiency <30%),the phage display-subcellular localization approach is highly efficiency in identifying surface protective antigens of bacteria.To evaluate the surface exposure,the defined proteins of E.rhusiopathiae were used for whole-cell ELISA and immunoblotting.A total of 12 protective antigens were defined(three of them have been reported),10 of which were screened by both phage libraries.Thesurface protein which were showed a high surface exposure or strong immunogenicity presents a high proportion of screening hits.Significantly,ADKPRVDTTTYN and NADKPTE,LQASAKTMHGTI and GAKWMSQ,AHRYIDAQIDRR and LALDRRD showed high similarity in the same amino acid regions(i.e.,possible epitopes)of Bml,Plp,and CwpB respectively,indicating that this is a highly effective procedure for protective antigen screening.Among the newly identified protective antigens of E.rhusiopathiae,the discovery of Bga and Da is surprising,since both are thought to be involved in metabolic regulation.Our results revealed that Bga conferred protection to 40% of mice against bacterial challenge and may be involved in E.rhusiopathiae pathogenesis.Da also conferred protection to 40% of mice against bacterial challenge and may present a higher surface-exposed in vivo relative to in vitro,suggesting that the antibody induced by Da may play an important role in immune protection against E.rhusiopathiae.Application of this novel approach can also provide additional insights into the function of bacterial surface antigens.