TEXT

Top Abstract Text References

Scrub typhus is characterized by fever, rash, eschar, pneumonitis, meningitis, and disseminated intravascular coagulation in some cases leading to circulatory collapse (10). It is caused by infection with Orientia tsutsugamushi, which belongs to the family Rickettsiaceae (21).

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). 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). Recent study has shown that antibody to Bor56 neutralizes oriental infection in vitro (17). The strong immune response of humans to this surface protein shows its potent immunogenicity (4, 6, 12, 14). As a result, Tsa has become the primary candidate for a genetically engineered scrub typhus vaccine. Since distinct determinants on this molecule could form the basis of a recombinant vaccine, determination of antigenicity and immunoaccessibility of epitopes should permit the rational selection of candidate domains. In an effort to identify strain-specific and cross-reactive epitopes of Tsa from strains Gilliam, Karp, Kato, and Boryong, we have generated a group of deletion fragments of the tsa gene encoding various regions of the protein. By using these constructs, we have identified domains which react with homotypic and heterotypic antibodies from the hyperimmunized mice.

Sera from hyperimmunized mice. Ten female BALB/c mice were immunized subcutaneously with O. tsutsugamushi as described previously (16). Three weeks after the third immunization, mice were bled and sera were prepared (3). Titers of antibody to O. tsutsugamushi and to MalE were examined (11, 12). Sera that showed a titer of antibody to a homotypic strain of more than 1:320 were used after heat inactivation by incubation at 56°C for 30 min.

Generation of Tsa mutants. To obtain the desired Tsa deletion (Tsa) mutants, parts of tsa were amplified by PCR, creating a series of fusion proteins that contain NH₂-terminal MalE fused with various lengths of coding sequences, as indicated in Fig. 1. tsa open reading frames of O. tsutsugamushi Gilliam, Karp, Kato, and Boryong were retrieved from the oriental genomic DNAs by PCR (12). Prokaryotic expression plasmids encoding truncated forms of Tsa were expressed in Escherichia coli XL1-Blue (Stratagene, La Jolla, Calif.). The nucleotide sequences of the 5' ends of the deletion constructs were determined by using malE primer (New England Biolabs, Beverly, Mass.). The first amino acids inferred from the 5' end of each deletion clone are shown in Fig. 1. Each of these expression clones was induced by the addition of isopropyl--D-thiogalactopyranoside (IPTG; Sigma, St. Louis, Mo.). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed as described previously (11, 12). The constructs encoded a fusion product that was clearly distinguishable on a Coomassie-stained gel (data not shown). MalE from the lysate of E. coli transformed by expression vector pIH821 was also analyzed. Figure 2A shows an immunoblot analysis of the constructs illustrated in Fig. 1 following the induced overexpression (19).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the fragments (1 to 8) of cloned tsa genes based on the bor56 nucleotide sequences and inferred amino acid sequences (GenBank accession no. L04956). The malE sequences of expression vector pIH821 are fused to portions of the fragments corresponding to the amino terminus and are not depicted. Numbers beside the fragments refer to amino acid residues of the translated tsa gene.

View larger version (87K): [in this window] [in a new window]   FIG. 2.   (A) Immunoblot of Tsa fusion proteins with sera from hyperimmunized mice. Induced fusion constructs were lysed, electrophoresed, transferred to nitrocellulose papers, and reacted with the indicated polyclonal sera (see below). Numbers indicate Tsa fragments shown in Fig. 1. (B) Densitometry analysis of the immunoblot. (C) Summary of immunoblotting analysis of sera from hyperimmunized mice with Tsa fragments. Black squares and gray squares indicate strongly positive and positive reactions (see the text), respectively. sGilliam, sKarp, sKato, and sBoryong, sera from mice immunized with Gilliam, Karp, Kato, and Boryong, respectively. G, P, T, and B, amino acid fragments derived from Gilliam, Karp, Kato, and Boryong, respectively.

Antibody responses to Tsa. The reactivities of the Tsa constructs with sera from hyperimmunized mice were analyzed after the immunostained bands were digitized (Fig. 2B). The images on the immunostained nitrocellulose membranes were digitized with a scanner (ScanJet 4100C; Hewlett-Packard, Boise, Idaho). The images were converted to gray scale. The densities of the bands were measured by using ScionImage (version beta 2; Scion Corporation, Frederick, Md.). The density values were assigned arbitrarily by ScionImage (Fig. 2B). The minimum value among the background gray scales was subtracted from the density values of the bands. Sera from the mice immunized with O. tsutsugamushi were analyzed for reactivity to MalE after proteins from pIH821-transformed E. coli were separated. Average values (AV) and standard deviations (SD) of the band densities derived from the antibody responses to MalE were calculated as described above. The AV of the sera from the mice immunized with Gilliam, Karp, Kato, and Boryong were 6.6, 6.3, 11.5, and 9.9, respectively. The SD were 2.2, 3.3, 2.3, and 2.3, respectively. The density values of the bands over the sum of the AV and the number obtained by multiplying the SD by 2 were considered positive reactions. The density values larger than the sum of the AV and the number obtained by multiplying the SD by 5 were regarded as strongly positive reactions.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   UPGMA dendrogram, derived from similarity coefficients, showing the relationships among O. tsutsugamushi strains based on the amino acid sequences of AD II and VD IV. Amino acid sequences were aligned and analyzed as described previously (18). The statistical validity of the trees was investigated by the bootstrap method of numerical resampling, using the SEQBOOT, PRODIST, NEIGHBOR, and CONSENS programs of the PHYLIP package (5). One hundred bootstrapped data sets of amino acid sequences for AD II and VD IV were generated. The distance matrices from each of the replicate data sets were calculated, and the tree was generated by successive clustering by using an average linkage method. The numbers at the forks indicate the number of times the group consisting of the species which are to the right of that fork occurred among the trees.