Abstract:
The inventive subject matter relates to a recombinant 110 kDa protein from  O. tsutsugamuchi,  Karp, Kato and Gilliam strains and for a DNA expression system containing DNA encoding the 110 kDa protein of  O. tsutsugamuchi.  The invention also relates to the use of these recombinant contructs in a formulation for the induction of a protective immune response against  O. tsutsugamuchi  invection using. The inventive subject matter also relates to a recombinant 110 kDa  O. tsutsugamuchi  protein or 110 kDa fragments for the production of antigen for use in immunodiagnosistic asssays for scrub typhus.

Description:
CROSS REFERENCE TO RELATED APPLICATION  
       [0001]     This application claims priority to U.S. Provisional application 60/563,447 filed Apr. 20, 2004. 
     
    
     SEQUENCE LISTING  
       [0002]     The disk Labeled “Cloning and expression of the full length 110 kDa antigen of  O. tsutsugamushi  to be used as a vaccine component against scrub typhus” containing the sequence listing file nc96,303.st25 is incorporated by reference. The file contains the same information that is provided in paper form as part of the application.  
       FIELD OF THE INVENTION  
       [0003]     This invention relates to the protection against infection of  Orientia tsutsugamushi.    
       DESCRIPTION OF PRIOR ART  
       [0004]     Scrub typhus infection is caused by the Gram-negative bacterium  Orientia tsutsugamushi.  It accounts for up to 23% of all febrile episodes in endemic areas of the Asia-Pacific region and can cause up to 35% mortality if left untreated [1,2]. Vaccines offer the potential of long-term prevention of morbidity and mortality from scrub typhus. They also obviate the difficulties posed by vector control and preventative chemoprophylaxis. The recent evidence of antibiotic resistance of  O. tsutsugamushi  further emphasizes the need of a scrub typhus vaccine [3,4,5]. Prior vaccine development efforts using the whole organism have suggested that a scrub typhus vaccine is possible. Immunization of volunteers with live vaccine in combination with tetracycline prophylaxis elicited immunity comparable to that of natural infection [6,7]. A polyvalent gamma irradiated vaccine that elicited some protection against heterologous serologic types was also demonstrated [8]. However due to the difficulties in mass production of purified  O. tsutsumagushi  and its instability upon storage, no useful product which meets today&#39;s FDA standards has been provided [9].  
         [0005]     Whole-organism vaccines have been previously developed and their protections have been short-lived and lack of cross strain protection. The major surface protein antigen, the variable 56-kDa protein which account for the antigenic variation, has been shown to induce protective immunity against the homologous strain but not the heterologous strains. The fact that other antigens, such as 110, 47 and 22 kDa have also been identified with high seroreactivity suggests that a combination of several of these antigens may provide better protection against various stains of  O. tsutsugamushi  infection [10].  
         [0006]     Although vaccination with a DNA construct or a recombinant protein of the major outer membrane protein 56 kDa antigen has been shown to provide protection against homologous challenge, complete cross protection from heterologous challenge has not been obtained. A minor 110 kDa antigen is also recognized by patient sera, suggesting it may provide additional protection against the  O. tsutsugamushi  infection. In order to develop a better and broadly protective vaccine candidate, we have successfully cloned the gene coding for the whole ORF of 110 kDa protein from major  O. tsutsugamushi  strains, including Karp, Kato and Gilliam into an expression system in order to generate a potential DNA vaccine candidate. Evaluation of the efficacy of the DNA constructs as potential vaccine formulations was also conducted in mice with or without either IL-2 or GM-CSF as adjuvant. Co-immunization with Karp containing DNA construct and pGM-CSF provided 60% protection whereas co-immunization with IL-2 afforded much less protection. These results suggest that the DNA expression system, alone or with GM-CSF, is useful in vaccine formulations against  O. tsutsugamushi  infection and as a prophylactic against scrub typhus.  
       SUMMARY OF THE INVENTION  
       [0007]     Accordingly, an object of this invention is a recombinant construct and expressed polypeptide possessing immunogenic regions.  
         [0008]     Another object of the invention is an expression system for expressing the  O. tsutsugamushi  110 kDa protein comprising cloning and amplifying the DNA sequence encoding the  O. tsutsugamushi  110 kDa protein and inserting and ligating the digestion product into a suitable expression system wherein the protein is expressed.  
         [0009]     Still another object of the invention is an immunogenic composition comprising a plasmid expressing the DNA sequence encoding an  O. tsutsugamushi  strain 110 kDa protein, wherein the protein is expressed and a plasmid expressing the DNA sequence encoding a IL-12 protein, wherein the protein is expressed and wherein an immune response is induced in a subject.  
         [0010]     Yet another object of the invention is an immunogenic composition comprising a plasmid expressing the DNA sequence encoding an  O. tsutsugamushi  strain 110 kDa protein, wherein the protein is expressed and a plasmid expressing the DNA sequence encoding a GM-CSF protein, wherein the protein is expressed and wherein an immune response is induced in a subject.  
         [0011]     Still another object of the invention the expression of the 110 kDa protein antigen in different host backgrounds of bacterial strains for use in different vaccine formulations against scrub typhus infection.  
         [0012]     Yet still another object of the invention is a vaccine formulation comprising one or more polypeptide sequences of the 110 kDa protein of  O. tsutsugamushi  with or without adjuvant.  
         [0013]     These and other objects, features and advantages of the present invention are described in or are apparent from the following detailed description of preferred embodiments. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0014]     The invention will be described with reference to the drawings, in which like elements have been denoted throughout by like reference numerals. The representation in each of the figures is diagrammatic and no attempt is made to indicate actual scales or precise ratios. Proportional relationships are shown as approximations.  
         [0015]      FIG. 1  shows the western blot confirmation of the expression of 110 kDa antigen from the  O. tsutsugamushi  strains Karp, Kato and Gilliam. Lane 1 is the molecular weight markers; lane 2 is the 110 kDa Kato strain protein inserted into VR 1020; lane 3 is the 110 kDa Karp strain protein inserted in VR 1020; lane 4 is the 110 kDa Gilliam strain protein inserted into VR 1020; lane 5 is the 110 kDa Gilliam strain inserted in VR 1012; lane 6 is VR 1020 alone; lane 7 is VR 1012 alone; lane 8 is the culture supernatant of 110 kDa Kato strain inserted into VR 1020; lane 9 is the culture supernatant of 110 kDa Karp strain inserted into VR 1020; lane 10 is the culture supernatant of 110 kDa Gilliam strain inserted into VR 1020; lane 11 is VR 1012 culture supernatant only; and lane 12 is VR 1020 culture supernatant only. 
     
    
     DETAILED DESCRIPTION  
       [0016]     There are still no FDA licensed vaccines available for scrub typhus. Previous vaccine candidates require bacterial antigen that must be purified by extremely labor intensive methods after first propagating the organism in specialized laboratories (BSL-3). The short coming of non-living protein vaccines is that it can not produce specific CD8+ T-cells, which is required for cellular immunity. Both humoral and cellular responses are likely to be required for the protection against intracellular pathogens such as human immunodeficiency virus,  Mycobacterium tuberculosis, Leishmania major  and  Plasmodium yoelii.  Wolff et al has shown that direct intramuscular inoculation of plasmid or naked DNA encoding several reporter genes could induce protein expression within the muscle cells [11]. DNA vaccines mimic the effects of live attenuated vaccines in their ability to induce both humoral and cellular responses, including class 1 restricted CD8+ T-cell responses, while eliminating some of the safety concerns associated with live vaccines. DNA vaccines are relatively easy to produce and can be used for protective antigen discovery. We have successfully cloned the gene coding for the whole ORF of 110 kDa protein from the Karp strain of  O. tsutsugamusi  into a VR1020 plasmid as the DNA vaccine (Kp 110 DNA) (12, 13). The move toward using DNA vaccines has the potential to shorten the time necessary for developing and fielding an effective polyvalent vaccine against scrub typhus. This is especially important because of the extensive antigen diversity in the 110 kDa protein antigen found among the various strains of  O. tsutsugamushi.  Here we disclose recombinant constructs from the 110 kDa gene of  O. tsutsugamushi.    
       EXAMPLE 1  
     Cloning And Expression of Recombinant 110 kDa Gene  
       [0017]     The open reading frame (ORF) of the 110 kDa gene of  O. tsutsugamushi  was obtained by polymerase chain reaction (PCR) amplification. The forward primer, SEQ ID No. 1, comprised the 5′ DNA sequence of 110 kDa ORF starting with the methionine initiation site but with an added kpn DNA restriction site at the 5′ end. Similarly, the reverse primer containing the stop codon of the ORF, contained in SEQ ID No. 2, was designed with a Kpn I site at its 5′ end. DNA template for the PCR reactions was obtained from DNA isolated from plaque-purified  O. tsutsugamushi  Karp strain grown in irradiated L929 cells [14]. The 110 kDa gene was amplified in a mixture of deoxynucleotide triphosphate, 1 mM of each primer, 1.5 U of Taq polymerase (Perkin-Elmer, CA) in 10 mM Tris-HCl buffer, pH 8.3, 1.5 mM MgCl 2 , and 50 mM KCl. The PCR reaction was started with 15 sec at 80° C., 4 min at 94° C., and followed by 30 cycles of 94° C. for 1 min, 57° C. for 2 min and 72° C. for 2 min. The last cycle was extended for 7 min at 72° C. Similar to the procedure used for the Karp strain, the ORF of the Kato and Gilliam strains was also amplified using the same forward primer as for Karp (SEQ ID No. 1) but reverse primers as in SEQ ID No. 3 for Kato and SEQ ID No. 4 for Gilliam. The sequence of the amplified Karp, Kato and Gilliam strain 110 kDa ORF is disclosed in SEQ ID No. 5, 6 and 7, respectively. When translated, these DNA sequences yield the amino acid sequence of SEQ ID No. 8, 9 and 10 for the Karp, Kato and Gilliam strains, respectively. Table 1 summarizes the sequences described.  
         [0018]     The above amplified PCR product containing the kpn (BioLab, MA) and BamH I (Life Technology, MD) sites were ligated to kpn digested VR 1020 expression vector to yield VR 1020/Karp, VR 1020/Kato or a VR 1020/Giliam strain 110 kDa protein expression system. The VR 1020/110 protein expression systems for Karp, Kato and Gilliam are designated pKp110, pKato110 and pGm110, respectively. Although VR 1020 was utilized, any plasmid or viral expression system can be used as long as polypeptide is expressed.  
         [0019]     Expression of the VR 1020/110 kDa expression systems are expressed in HEK 293 cell lines. Growing cultures of HEK 293 cell line containing these plasmids are then harvested and the cell culture fluid and cell lysate analyzed by western blotting using specific anti-110 kDa antiserum as a probe to evaluate expression of the 110 kDa ORF. As shown in  FIG. 1 , analysis of expressed product yields a full length 110 kDa moiety in both the culture fluid and cell lysate ( FIG. 1 ).  
                           TABLE 1                                   SEQ ID No.   Description                           SEQ ID No. 1   Forward PCR primer pKp110           SEQ ID No. 2   Reverse PCR primer pKp110           SEQ ID No. 3   Reverse PCR primer pKato110           SEQ ID No. 4   Reverse PCR primer pGm110           SEQ ID No. 5   Karp 110 DNA sequence           SEQ ID No. 6   Kato 110 DNA sequence           SEQ ID No. 7   Gilliam 110 DNA sequence           SEQ ID No. 8   Amino acid sequence Karp 110           SEQ ID No. 9   Amino acid sequence Kato 110           SEQ ID No. 10   Amino acid sequence Gilliam 110                      
 
         [0020]     Based on the above studies, the recombinant constructs can be utilized to induce a protective immune response in humans. The immunizing composition will be composed of: 
        a. a plasmid, such as VR 1020, containing the DNA sequence encoding the entire or a fragment of the  O. tsutsugamushi  110 kDa protein, wherein said protein is expressed; and     b. a plasmid, such as VR 1020, containing the DNA sequence encoding a cytokine adjuvant, wherein said cytokine adjuvant is expressed and wherein an immune response is induced. The cytokine adjuvant is either IL-12 or GM-CSF.        
 
         [0023]     The DNA sequence inserted into the plasmid is either the entire or fragment of  O. tsutsugamushi  110 kDa protein derived from one or more of the  O. tsutsugamushi  strains Karp, Kato and Gilliam. Furthermore, the sequence inserted is all or a portion of the DNA sequence of SEQ ID No. 5, 6 and 7 and which encodes the entire or a fragment of one or more of polypeptide sequences of SEQ ID No. 8, 9 and 10.  
         [0024]     The method of inducing an immune response comprises the following steps: 
        a. administering a priming dose comprising 2-10 mg per dose each of one or more plasmids containing a DNA sequence encoding the entire or fragment of the110 kDa protein from one or more  O. tsutsugamuchi  strains selected from the group consisting of Karp, Kato and Gilliam, wherein said protein is expressed and a plasmid containing the DNA sequence encoding a cytokine adjuvant, wherein said cytokine adjuvant is expressed;     b. admininistering 1 to 4 boosting doses with the first boosting dose at least 1 week after said priming dose.        
 
         [0027]     The boosting dose contains all or a fragement of one or more  O. tsutsugamuchi  DNA sequences SEQ ID No. 5, 6 and 7 encoding the Karp, Kato and Gilliam 110 kDa polypeptides SEQ ID No. 8, 9, and 10 from  O. tsutsugamuchi.  The boosting DNA sequence, however, is from the same strain as in the priming dose. The boosting dose also can include a plasmid expressing a DNA sequence encoding a cytokine adjuvant such as IL-12 or GM-CSF.  
       EXAMPLE 2  
     Use of Kp 110 DNA as a Vaccine Candidate in Mouse Model  
       [0028]     The ability of the pKp110 to elicit a protective immune response, murine studies were conducted using these constructs as immunogen. Female Swiss outbred CD-1 mice (Charles River Laboratories, Wilmington, Mass.), 18-24 g, were used throughout the study. Mice were immunized intramuscularly with 28 g×½″ needle at the two thighs 25 ul/site, total of 50 ul containing different amount of Karp 110DNA. Mice were challenged with the lethal dose of 1000×LD 50  of Karp in 0.2 ml of Snyder&#39;s 1 buffer four weeks after one immunization. Date of onset of disease and date of death were recorded for individual mice. The morbidity and mortality were monitored at least twice a day for 21 days post-challenge.  
         [0029]     The protective efficacy of pKp 110 DNA against challenge in mouse model is summarized in Table 1 and Table 2. As shown in Table 1, pKp110 demonstrated a protective efficacy with IL-2 or GM-GSF that was significantly better than that of pKp56, which is the VR1012 expression vector containing the 56 kDa protein construct of  O. tsutsugamushi.  However, pKp 110 was equivalent but slightly less efficacious than pKp47, which is the VR 1020 vector containing the 47 kDa recombinant construct of  O. tsutsugamushi.  However, a likely advantage of using the 110 kDa construct verses the 47 kDa construct is because of the potential for induction of an autoimmune response by the 47 kDa immunogen. This possibility is predicated based on the homology of a large region of the 47 kDa DNA sequence (15) with the eukaryotic trypsin-like gene (16, 17).  
                                                                                 TABLE 2                           Protection of Mice immunized with pKarp110 using pIL-12 (10 ug) as       adjuvant                % Survival                Immunogen   Exp 1(10 3 )   Exp 2(10 5 )   Exp 3(10 3 )                        1.   PBS (IL-12 only)   1/12   8.3%                        2.   p1012, 100 ug   1/10   10%   0/10   0%   0/10   0%       3.   p1020, 100 ug   1/9    11%   1/10   10%   1/10   10%       4.   pKp110 (no IL-12),           1/10   10%   4/10   40%           100 ug       7.   pKp56, 100 ug   5/12   42%   0/10   0%   2/10   20%       8.   pKp56, 50 ug           1/10   10%   2/10   20%       9.   pKp47, 100 ug   6/10   60%   2/10   20%   7/10   70%       10.   pKp47, 50 ug           4/10   40%   6/10   60%       11.   pKp110/pKp56, 50 ug   6/10   60%   3/10   30%   7/10   70%           each       12.   pKp110/pKp47, 50 ug           9/10   90%   8/10   80%                  
 
         [0030]    
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                   
               
               
                 Protection of Mice Immunized with pKarp110 using pGM-CSF (10 ug) 
               
               
                 as the adjuvant 
               
             
          
           
               
                   
                 % Survival 
               
             
          
           
               
                   
                 Immunogen 
                 Exp 1 (10 3 ) 
                 Exp 2 (10 5 ) 
                 Exp 3 
               
               
                   
                   
               
             
          
           
               
                 1. 
                 PBS (GMCSF only) 
                 3/12 
                 25% 
                   
                   
                   
                   
               
               
                 2. 
                 p1012, 100 ug 
                 0/12 
                 0% 
                 0% 
                 0/10 
               
               
                 3. 
                 p1020, 100 ug 
                 1/10 
                 10% 
                 0% 
                 0/10 
               
               
                 4. 
                 pKp110 (no GMCSF), 
               
               
                   
                 100 ug 
               
               
                 7. 
                 pKp56, 100 ug 
                 3/12 
                 25% 
                 3/10 
                 30% 
               
               
                 8. 
                 pKp56, 50 ug 
                   
                   
                 1/10 
                 20% 
               
               
                 9. 
                 pKp47, 100 ug 
                 10/10 
                 100% 
                 7/10 
                 70% 
               
               
                 10. 
                 pKp47, 50 ug 
                   
                   
                 10/10  
                 100% 
               
               
                 11. 
                 pKp110/pKp56, 50 ug 
                 4/10 
                 40% 
                 7/10 
                 70% 
               
               
                   
                 each 
               
               
                 12. 
                 pKp110/pKp47, 50 ug 
                   
                   
                 8/10 
                 80% 
                 12/15 
                 80% 
               
               
                   
                 each 
               
               
                 13. 
                 pKp110/pKp47, 25 ug 
                   
                   
                   
                   
                  9/15 
                 60% 
               
               
                   
                 each 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 3  
     Antigen Reagent for Scrub Typhus Assays and Subunit Vaccines  
       [0031]     The recombinant 110 kDa  O. tsutsugamushi  antigen, because of its immunoreactivity, has significant utility as a diagnostic antigen in immunoassays for scrub typhus. The recombinant antigen, because of its high-level of immunoreactivity to patient sera, is well suited as a standardized antigen for assays designed for the detection of prior infection by  O. Isutsugamushi  and diagnosis of scub typhus. Recombinant 110 kDa antigen can be incorporation into any antibody-based assay including enzyme-linked immunosorbent assays and rapid flow immunoassays. The antigens are easily recombinantly expressed using any expression system, including pET 24 and are thus capable of standardized production quality.  
         [0032]     An example of an expression system for recombinant expression of  O. tsutsugamuchi  110 kDa antigen is the construction of the pET 24d/ O. tsusugamuchi  vector is constructed by first introducing DNA encoding for the  O. tsutsugamuchi  10 kDa protein into the pET 24d vector.  
         [0033]     An expression system encoding 110 kDa antigen can be constructed by inserting either DNA encoding the entire 110 kDa protein or fragements of the gene or DNA sequences encoding a portion of the 110 kDa gene. In this example, either  O. tsutsugamuchi  Karp, Kato or Gilliam strain DNA for fragment A, which encodes for Gly 140 to Asn 587 of the 110 kDa protein or fragment B, encoding for Val 507 to Asn 903 of the 110 kDa protein, is inserted into the pET24d vector. The DNA sequence of Karp, Kato and Gilliam strains fragment A is SEQ ID No. 11, 13 and 15 respectively. These sequences encode the Karp, Kato and Gilliam polypeptide sequences SEQ ID No. 17, 19, and 21, respectively. The DNA sequence for Karp, Kato and Gilliam strains for fragment B is SEQ ID No. 12, 14 and 16 which encodes for the Karp, Kato and Gilliam polypeptides sequences SEQ ID No.18, 20 an 22, respectively. The  O. tsutsugamuchi  fragment and its associated SEQ ID numbers are summarized in Table 4.  
         [0034]     Each of the recombinant  O. tsutsugamuchi  110 kDa polypeptides are similarly constructed. For example, the fragments A or B of the Karp strain is produced by amplifying the fragment from native DNA with PLATINUM Taq DNA POLYMERASE HIGH FIDELITY® (Invitrogen, Carlsbad, Calif.) using genomic DNA of  O. tsutsugamuchi  karp strain as template. The forward primer for fragment A was SEQ ID number 23 and the reverse primer was SEQ ID No. 24. The resulting PCR product was then inserted between the NcoI and EcoRI sites of the pET24d plasmid. The resulting plasmid pET24d-110A Karp encodes the A fragment (Gly-140 to Asn-587). For fragment B, the forward and reverse PCR primers were SEQ ID No. 25 and 26, respectively. The fragment B (Val-507 to Asn-903) sequence was inserted into pET24d as for fragment A. The sequence of both constructed plasmids (pET24d-110A Karp and pET24d- 110B Karp) was verified by sequencing.  
         [0035]     The pET 24 vectors containing the 110 kDa fragment A and B proteins were expressed in  E. coli  BL21(DE3) bacteria. Cells were grown in L-broth containing 50 μg/ml kanamycin at 37° C. to an O.D. 600  0.8 at which time 1PTG (1 mM) was added. The culture was then incubated with shaking for 4 hrs at 37° C. Cells were harvested and weighted (about 5.5 g of wet cells per liter culture). The cell pellets were re-suspended in 7 volume of buffer A (20 mM Tris-HCI, pH 8.0, 5 mM EDTA), and lysed. The cell lysate was cleared by first centrifugation at 6,000 rpm (IEC MultiRF rotor) for 10 min then a second centrifugation at 9,600 rpm (the same rotor) for 30 min. The 110 kDa antigen fragments were then precipitated by adding solid ammoninm sulfate to the lysate to 30% saturation (0.164 g/ml) for fragment A and 40% saturation (0.226 g/ml) for fragment B. After centrifugation at 9,600 rpm for 30 min at 4° C., the protein pellet was re-suspended with one-seventh volume of buffer A. Subsequent to resuspension in Tris buffer, fractions A and B were purified through a gel filtration column (ZORBAX Bio Series GF-450™, Agilent Technology, Palo Alto, Calif.).  
         [0036]     The  O. tsutsugamushi  peptides can be utilized either alone or in combinantion with other  O. tsutsugamushi  fragment antigens in immunodiagnostic assays comprising the following steps: 
        1. Microtiter plates with 96 wells were coated with 0.3 μg/well of any or all of the recombinant proteins represented by SEQ ID No. 17-22 and stored in 4° C. for 2 days.     2. Plates are washed ×3 with wash buffer (0.1% Tween-20 in PBS).     3. Plates are blocked with 200 μl/well of blocking buffer (5% skim milk in wash buffer) for 45 minutes and then rinsed three times.     4. Sera is diluted in blocking buffer and 100 μl/well is added and incubated for 1 to 2 hours.     5. Plates are washed three times with wash buffer.     6. Plates are then incubated with 100 μl/well of enzyme-labeled (e.g. peroxidase) anti-human immunoglobulin for 1 hour.     7. The plates are washed three times with wash buffer.     8. Substrate is added to the wells and read after 15 to 30 minutes.        
 
         [0045]     A standard curve is constructed by conducting the above ELISA procedures with the recombinant proteins but utilizing a range of concentrations of specific antibody to  O. tsutsugamuchi.  The extent of measured binding of patient serum antibody is compared to a graphic representation of the binding of the  O. tsutsugamuchi -specific antibody concentrations.  
         [0046]     Sensitivity of antibody-based assays, such as ELISA, can be enhanced by substituting the enzyme-substrate step with a molecular detection method. An example of a molecular method employed is the amplification of circular DNA by rolling circle amplification (RCA). In RCA, antibody specific to  O. tsutsugamuchi  is conjugated with a single stranded DNA primer comprising the following steps: 
        a. 1 mg of sulfo-GMBS powder was added to 4 mg of antibody F(ab′) 2  in 1 ml, in the dark, for 30 minutes at 37° C., followed by 30 minutes at room temperature;     b. 2 ml of phosphate buffered saline (PBS) was added to the reaction mixture from a;     c. the reaction mixture in b, above, was applied unto a preequilibrated Presto Desalting Columns (Pierce Biotechnology, Inc, Rockford, Ill.);     d. the applied material was eluted with PBS and the eluted fractions monitored by absorbance at 280 nm;     e. pooled fractions containing maleimide-acitivated antibody was concentrated and stored at 4° C. in the dark until used;     f. activated DNA was prepared by res-suspending 0.44 mg of thio-DNA (C6 S-S®) (MWG-Biotech Inc, High Point, N.C.) in 150 μl TE buffer with 15 μl of 1 M DTT and incubated at room temperature for 30 minutes;     g. the DTT was removed from the mixture of step g by applying the mixture to a G-50 micro column and spinning the column at 735× g for 2 minutes;     h. the activated antibody and activated thio-DNA was then mixed and the mixture incubated in the dark at room temperature for 1 hour then overnight at 4° C.;     i. product from step h was analyzed by gel electrophoresis.        
 
         [0056]     RCA reactions were undertaken the method comprising the following steps: 
        a. mix together on ice 5 nM of primer-conjugated antibody, 10 nM circular DNA, 200 ng of  E. coli,  single-stranded DNA binding protein (SSB), 13 units of T7 Sequenase and 0.4 mM each of dATP, dCTP, dGTP, 0.3 mM dTTP and 0.1 mM FITC-dUTP in 25 μl of reaction buffer at pH 7.9 containing 20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate and 1 mM DTT;     b. incubate the mixture of step at 37° C. for up to 30 minutes;     c. RCA products are then analyzed analyze the products by measuring fluorescence incorporation of DNA product.        
 
         [0060]     As alternative to RCA, PCR can be utilized using a primer complimentary to the antibody-conjugated DNA, made as described for RCA. Amplification is conducted by utilizing a DNA primer complementary to a template sequence contained on the conjugated DNA.  
         [0061]     In addition to immunoassays, the recombinant amino acid sequences can be utilized to induce an immune response, as in a vaccines against  O. tsutsugamushi  infection. A prophetic example of the use of the amino acid sequences comprises the following steps: 
        a. administering a priming dose comprising 50 μg to 2 mg per dose of one or more of the entire or fragment of a recombinant polypeptide encoded an amino acid sequence selected from the group consisting of SEQ ID No. 8, 9 and 10; and     b. administering 1 to 4 boosting doses with the first boosting dose at least 1 week after priming dose comprising 50 μg to 2 mg per dose of one or more of the entire or fragment of a recombinant polypeptide encoded by an amino acid sequence selected from the group consisting of SEQ ID No. 8, 9, and 10, wherein an immune response is elicited.        
 
         [0064]     In the above example for inducing an immune response, a cytokine adjuvant can be included either in the administration of the priming or boosting doses or upon both the priming and boosting administrations of the polypeptides. The cytokine adjuvant can be any cytokine including IL-12 or GM-CSF.  
                           TABLE 4                                   SEQ ID No.   Description                           SEQ ID No. 11   DNA fragment A Karp strain           SEQ ID No. 12   DNA fragment B Karp strain           SEQ ID No. 13   DNA fragment A Kato strain           SEQ ID No. 14   DNA fragment B Kato strain           SEQ ID No. 15   DNA fragment A Gilliam strain           SEQ ID No. 16   DNA fragment B Gilliam strain           SEQ ID No. 17   Amino acid sequence fragment A Karp strain           SEQ ID No. 18   Amino acid sequence fragment B Karp strain           SEQ ID No. 19   Amino acid sequence fragment A Kato strain           SEQ ID No. 20   Amino acid sequence fragment B Kato strain           SEQ ID No. 21   Amino acid sequence fragment A Gilliam strain           SEQ ID No. 22   Amino acid sequence fragment B Gilliam strain                      
 
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         [0082]     Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.