Identification of cross-reactive epitopes on the conserved 47-kilodalton antigen of Orientia tsutsugamushi and human serine protease

Abstract

Orientia tsutsugamushi is the causative agent of scrub typhus. One of the protein antigens of this species, the conserved 47-kDa protein (HtrA), has been shown to induce an antibody response in patients and can provide protective immunity against live challenge by Orientia in mice. Pepscan experiments identified many peptide epitope clusters in different parts of this protein. The majority of the most reactive epitopes are located at the C terminus of the protein (from amino acid 333 to amino acid 430). Protein sequence analysis revealed that the 47-kDa protein contains a trypsin domain and has sequence homology to human serine protease HtrA1 (hHtrA1). As the 47-kDa protein is a potential vaccine candidate and its ability to induce autoimmunity is a concern, the reactivity of scrub typhus patient sera with purified recombinant 47-kDa and hHtrA1 proteins was tested. A significant percentage (>20%) of scrub typhus patient sera reacted strongly with recombinant hHTRA1 and two of the antigenic polypeptide epitopes in hHtrA1. These findings suggest that the safety of the full-length 47-kDa antigen as a vaccine candidate is a significant issue due to its cross-reactivity with a human protein, which may also contribute to autoimmune responses or enhanced pathology in some scrub typhus patients.

FIG. 1.

The 47-kDa antigen of O. tsutsugamushi has significant sequence homology to hHtrA1 in a 151-amino-acid region. Red type indicates the three amino acids (histidine, aspartic acid, and serine) located in the catalytic site of human HtrA1; asterisks indicate identical amino acids; colons indicate semiconserved amino acids; and periods indicate similar amino acids.

FIG. 2.

ELISA results for 480 scrub typhus patient sera with rhHtrA1. The cutoff values are 0.117 for rhHtrA1 and 0.158 for r47b (means for 82 negative controls plus 2 standard deviations). (A) Relationship of reactivity (as reflected by the optical density determined by ELISA) to the number of days after the onset of fever. The x axis indicates the number of days after the onset of illness, and the y axis indicates the optical density at 405 nm. (B) Time course of reactivity of patient MAK116 sera collected at days 3, 10, 19, 27, 31, and 64 with the recombinant 47-kDa (▪) and rhHtrA1 (▴) proteins. The symbols indicate the averages of two experiments, and the error bars represent ranges. O.D., optical density.

FIG. 3.

ELISA profiles for five patient sera and one normal serum with overlapping synthetic decapeptides encompassing the 47-kDa antigen of O. tsutsugamushi Karp. The five MAK numbers indicate five different patients. The amino acid position for each decapeptide is the first amino acid. Each successive decapeptide begins four residues after the preceding peptide.

FIG. 4.

ELISA results for 41 rhHtrA1-positive patient sera with synthetic peptides. P216-236 (A) and P285-308 (B) were tested with 41 rhHtrA1-positive sera from serial bleeding of 26 scrub typhus patients. The cutoff values for positive results are 0.221 and 0.237 (means of 10 negative controls plus 2 standard deviations) for P216-236 (A) and P285-308 (B), respectively. O.D., optical density.

FIG. 5.

Time course of antibody responses of two patients to hHtrA1 synthetic peptides. P216-236 (A) and P285-308 (B) were tested with sera from serial bleeding of patient MAK116 (▪) collected at days 3, 10, 19, 27, 31, and 64 and patient MAK131 (▴) collected at days 7, 14, 21, 28, 35, 71, and 96. The cutoff values for positive results are 0.221 and 0.237 (means of 10 negative controls plus 2 standard deviations) for P216-236 and P285-308, respectively. The symbols indicate the averages of two experiments, and the error bars represent ranges. O.D., optical density.