Mapping of antigenic determinant regions of the Bor56 protein of Orientia tsutsugamushi

Abstract

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.

Prokaryotic expression and immunogenicity of 56‐kDa protein of Orientia tsutsugamushi strain Karp

“…56-kDa protein, which was predicted to be hydrophobic and embedded in the membrane and showed no reactivity with human IgG and IgM antibodies (23). Previous reports have shown that 56-kDa protein was always produced in the form of inclusion body in E. coli (15)(16)(17)(18)(19)(20).…”

Inhibition ofOrientia tsutsugamushiinfection by a truncated recombinant 56-kDa outer membrane protein

“…Because the functional domain analysis of 56kDa protein was accomplished, the truncated 56‐kDa proteins were made for diagnostic tool and vaccine candidates, which were very effective. We then tried to make truncated proteins from a prior study (Seong et al. 1997b).…”

“…Although various truncated 56‐kDa proteins with deletion of both N‐ and C‐term domain differ in their infection ability, they share many structural features with other truncated 56‐kDa proteins (Ohashi et al. 1992; Seong et al. 1997a,b).…”

Homotypic and Heterotypic Antibody Responses to a 56-Kilodalton Protein of Orientia tsutsugamushi

“…The mechanisms responsible for protective immunity of O. tsutsugamushi-infected humans may involve both humoral and cell-mediated immunity (1, 2, 7-9, 13, 15-17, 20). Type-specific antigen (Tsa), a 56-kDa protein of O. tsutsugamushi, is a surface-exposed (22), major integral membrane protein (19). The immune responses to Tsa are important in preventing infection (16,17).…”

“…The immune responses to Tsa are important in preventing infection (16,17). Animals immunized with Tsa develop both humoral and cellular immune responses to O. tsutsugamushi markedly (16,17,19). Mice immunized with recombinant Bor56, one of the Tsa, were protected from challenge with the homotype of O. tsutsugamushi (16).…”