Kinetics and magnitude of antibody responses against the conserved 47-kilodalton antigen and the variable 56-kilodalton antigen in scrub typhus patients

Abstract

Western blot analysis of Orientia tsutsugamushi whole-cell lysates with scrub typhus patient sera has identified at least five protein antigens of O. tsutsugamushi with molecular sizes of 22 kDa, 47 kDa, 56 kDa, 58 kDa, and 110 kDa. In this study, sera from serial bleedings of 108 patients were used to study the kinetics and the magnitude of specific antibody responses against the 47-kDa and 56-kDa antigens. Recombinant protein of the conserved 47-kDa antigen (r47b) or a mixture of truncated 56-kDa antigen (r56s) from three prototype strains was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). Our results showed that 76% and 93% of these patients had elevated IgM and IgG against r47b, respectively, and 98% and 100% had elevated IgM and IgG against r56s, respectively. The kinetics of antibody responses against r47b and r56s can be grouped into three patterns. In the first type of response, IgM and IgG against r47b and r56s appeared about the same time. The IgM and IgG titers against r56s were much higher than those against r47b. In the second type of response, induction of IgM appeared to be similar to that in the first type. The major difference to the first type is that the IgG titers against r47b were induced at least 1 week later than those against the r56s. The third type showed strong IgG responses against both r47b and r56s, and low or no IgM responses indicated a secondary infection. This is the first systematic investigation of antibody response kinetics against the conserved 47-kDa antigen versus the variable 56-kDa antigen in scrub typhus patients.

Fig. 1.

IgM ELISA results for 437 scrub typhus patient serum samples with recombinant antigens. Specific IgM against r47b (A) and r56s (B) were tested with 437 patient serum samples from serial bleeding of 108 scrub typhus patients. The x axis indicates the number of days after the onset of illness, and the y axis indicates the optical density (O.D.) at 405 nm. The cutoff values are 0.182 for r47b and 0.330 for r56s (mean of 125 negative controls plus 2 standard deviations).

Fig. 2.

IgG ELISA results for 437 scrub typhus patient serum samples with recombinant antigens. Specific IgG against r47b (A) and r56s (B) were tested with 437 patient serum samples from serial bleeding of 108 scrub typhus patients. The x axis indicates the number of days after the onset of illness, and the y axis indicates the optical density (O.D.) at 405 nm. The cutoff values are 0.158 for r47b and 0.216 for r56s (mean of 125 negative controls plus 2 standard deviations).

Fig. 3.

Time course of antibody responses of two typical type I patients to r47b and r56s antigens. The serum samples of patient MAK135 (circle) were collected at days 3, 10, 17, 24, 31, 69, and 94, and the serum samples of patient MAK229 (square) were collected at days 5, 17, 26, 49, and 69. IgM (A) and IgG (B) specific for r47b (black) or r56s (red) in patient sera were measured by ELISA. The cutoff values are the same as in Fig. 1 and 2. The symbols represent the averages of two experiments, and the error bars represent ranges. O.D., optical density.

Fig. 4.

Time course of antibody responses of two typical type IIa patients to r47b and r56s antigens. The serum samples of patient MAK217 (circle) were collected at days 3, 12, 17, 26, and 52, and the serum samples of patient MAK240 (square) were collected at days 3, 10, 16, 25, and 63. IgM (A) and IgG (B) specific for r47b (black) or r56s (red) in patient sera were measured by ELISA. The cutoff values are the same as in Fig. 1 and 2. The symbols represent the averages of two experiments, and the error bars represent ranges. O.D., optical density.

Fig. 5.

Time course of antibody responses of two typical type IIb patients to r47b and r56s antigens. The serum samples of patient MAK142 (circle) were collected at days 3, 10, 17, 23, and 88, and the serum samples of patient MAK153 (square) were collected at days 3, 17, 24, 30, and 76. IgM (A) and IgG (B) specific for r47b (black) or r56s (red) in patient sera were measured by ELISA. The cutoff values are the same as in Fig. 1 and 2. The symbols represent the averages of two experiments, and the error bars represent ranges. O.D., optical density.

Fig. 6.

Time course of antibody responses of two typical type III (the secondary infection) patients to r47b and r56s antigens. The serum samples of patient MAK132 (circle) were collected at days 7, 14, 21, 28, and 69, and the serum samples of MAK168 (square) were collected at days 3, 10, 24, and 68. IgM (A) and IgG (B) specific for r47b (black) or r56s (red) in patient sera were measured by ELISA. The cutoff values are the same as in Fig. 1 and 2. The symbols represent the averages of two experiments, and the error bars represent ranges. O.D., optical density.