MAPPING OF ANTIGENIC DETERMINANT REGIONS OF THE BOR56 PROTEIN OF ORIENTIA-TSUTSUGAMUSHI

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprote ctive antigen and is the target molecule of neutralizing antibodies. T his antigen is recognized by almost all of the serum antibodies produc ed by patients in the convalescence phase of scrub typhus. We expresse d the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-b inding domains were characterized by using patient sera, mouse monoclo nal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the ant ibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-ter minal domain of Bor56 is not exposed on the surface of the molecule. H uman immunoglobulin M (IgM) antibodies predominantly bound to antigeni c domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 32 8). Human IgG antibodies also showed preferential binding to AD L. The epitope recognized by strain-specific MAb (K14) or group-specific MAb (K157) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, e licited by immunization with deletion mutants, consistently bound to A D HU. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did n ot elicit an antibody response in C3H/HeDub mice. A model of the antig enic structure of Bor56 is presented and discussed. These results sugg est that antigenic fragments from AD I and AD III are useful in the in duction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the n eutralizing-antibody responses generated during rickettsial infections . They also provide potential peptide substrates for diagnostic assays and vaccine strategies.