WO2006078271A2

WO2006078271A2 - Cloning and expression of the full length 110 kda antigen of orientia tsutsugamushi to be used as a vaccine component against scrub typhus

Info

Publication number: WO2006078271A2
Application number: PCT/US2005/013255
Authority: WO
Inventors: Wei-Mei Ching; Chien-Chung Chao
Original assignee: US Department of Navy
Current assignee: US Department of Navy
Priority date: 2004-04-20
Filing date: 2005-04-19
Publication date: 2006-07-27
Anticipated expiration: 2006-10-20
Also published as: WO2006078271A3

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

CLONING AND EXPRESSION OF THE FULL LENGTH 1 10 KDA ANTIGEN OF ORIENTIA TSUTSUGAMUSHI TO BE USED AS A VACCINE COMPONENT AGAINST SCRUB TYPHUS

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional application 60/563,447 filed April 20, 2004.

SEQUENCE LISTING

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 This invention relates to the protection against infection of Orientia tsutsugamushi.

DESCRIPTION OF PRIOR ART

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's FDA standards has been provided [9].

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 1 10, 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].

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 1 10 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 1 10 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

Accordingly, an object of this invention is a recombinant construct and expressed polypeptide possessing immunogenic regions. Another object of the invention is an expression system for expressing the O. tsutsugamushi HOkDa protein comprising cloning and amplifying the DNA sequence encoding the O. tsutsugamushi 11OkDa protein and inserting and ligating the digestion product into a suitable expression system wherein the protein is expressed.

Still another object of the invention is an immunogenic composition comprising a plasmid expressing the DNA sequence encoding an O. tsutsugamushi strain 1 1OkDa 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.

Yet another object of the invention is an immunogenic composition comprising a plasmid expressing the DNA sequence¹ encoding an O. tsutsugamushi strain HOkDa 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.

Still another object of the invention the expression of the 1 10 kDa protein antigen in different host backgrounds of bacterial strains for use in different vaccine formulations against scrub typhus infection.

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. 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

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.

Figure 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 1 10 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 1 1 is VR 1012 culture supernatant only; and lane 12 is VR 1020 culture supernatant only.

DETAILED DESCRIPTION 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 [H]. DNA vaccines mimic the effects of live attenuated vaccines in their ability to induce both humoral and cellular responses, including class I 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 VRl 020 plasmid as the DNA vaccine (Kp 110DNA) (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

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 1 10 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 MgCl², and 50 mM KCl. The PCR reaction was started with 15 sec at 80⁰C, 4 min at 94°C, and followed by 30 cycles of 94⁰C for 1 min, 57⁰C for 2 min and 72⁰C for 2 min. The last cycle was extended for 7 min at 72⁰C. 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.

The above amplified PCR product containing the kpn (BioLab, IVlA) 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 1020/1 10 protein expression systems for Karp, Kato and Gilliam are designated pKpl 10, pKatol 10 and pGml 10, respectively. Although VR 1020 was utilized, any plasmid or viral expression system can be used as long as polypeptide is expressed.

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 1 10 kDa ORF. As shown in Figure 1, analysis of expressed product yields a full length 110 kDa moiety in both the culture fluid and cell lysate (Figure 1).

Table 1

SEQ ID No. Description

SEQ ID No. 1 Forward PCR primer pKpl 10

SEQ ID No. 2 Reverse PCR primer pKpl 10

SEQ ID No. 3 Reverse PCR primer pKatol 10

SEQ ID No. 4 Reverse PCR primer pGml 10

SEQ ID No. 5 Karp 1 10 DNA sequence

SEQ ID No. 6 Kato 1 10 DNA sequence

SEQ ID No. 7 Gilliam 1 10 DNA sequence

SEQ ID No. 8 Amino acid sequence Karp 1 10

SEQ ID No. 9 Amino acid sequence Kato 110

SEQ ID No. 10 Amino acid sequence Gilliam 110

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 VK 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.

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.

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 thel 10 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.

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 Ϊ^'TO^' D^'NA as a vaccine candidate in mouse model.

The ability of the pKpl 10 to elicit a protective immune response, murine studies were conducted using these constructs as immunogen. Female Swiss outbred CD-I mice (Charles River Laboratories, Wilmington, MA), 18-24 g, were used throughout the study. Mice were immunized intramuscularly with 28 g x 'A" needle at the two thighs 25 ul/site, total of 50 ul containing different amount of Karp 11 ODNA. Mice were challenged with the lethal dose of 1000 x LD₅₀ of Karp in 0.2 ml of Snyder's I 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.

The protective efficacy of pKp 1 10 DNA against challenge in mouse model is summarized in Table 1 and Table 2. As shown in Table 1, pKpl 10 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 pKarpl 10 using pIL-12 (10 ug) as adjuvant Immunogen % Survival

Table 3

Protection of Mice Immunized with pKarpl 10 using pGM-CSF (10 ug) as the adjuvant

Immunogen % Survival

Exp 1 (10³) EXD 2 (10⁵) Exp 3

1. PBS (GMCSF only) 3/12 25% 2. plO12, 100 ug 0/12 0% 0% 0/10 3. pl020, 100 ug 1/10 10% 0% 0/10

1 1. pKpl 10/pKp56, 50 ug each 4/10 40% 7/10 70% tpfiCp! NM≠S≠% M ii <ss§fe m@ ■IMS SW

Bio , ≠&≠ IM0M% W> m ga§to TIMS u^pc

Example 3 Antigen reagent for Scrub typhus assays and subunit vaccines The recombinant 1 10 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. tsutsugamushi 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.

An example of an expression system for recombinant expression of O. tsutsugamuchi 110 kDa antigen is the construction of the pET 24d/6>. tsutsugamuchi vector is constructed by first introducing DNA encoding for the O. tsutsugamuchi 1 10 kDa protein into the pET 24d vector. An expression system encoding IWkJJa antigen can be constructed by inserting either DNA encoding the entire HOkDa protein or fragements of the gene or DNA sequences encoding a portion of the 1 10 kDa gene. In this example, either O. tsutsugamuchi Karp, Kato or Gilliam strain DNA for fragment A, which encodes for GIy 140 to Asn 587 of the 110 kDa protein or fragment B, encoding for VaI 507 to Asn 903 of the 1 10 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.

Each of the recombinant O. tsutsugamuchi 1 10 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, CA) 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 Ncol and EcoRI sites of the pET24d plasmid. The resulting plasmid pET24d-l 1OA Karp encodes the A fragment (GIy- 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-l 1OA Karp and pET24d-l 1OB Karp) was verified by sequencing.

The pET 24 vectors containing the 1 10 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 37⁰C to an O.D.βoo 0.8 at which time IPTG (1 mM) was added. The culture was then incubated with shaking for 4 hrs at 37⁰C. Cells were harvested and weighted (about 5.5g of wet cells per liter culture')". The cell ^"peϊϊefs were re-suspended in 7 volume of buffer A (20 mM Tris-HCl, 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 1 10 kDa antigen fragments were then precipitated by adding solid ammoninm sulfate to the lysate to 30% saturation (0.164g/ml) for fragment A and 40% saturation (0.226g/ml) for fragment B. After centrifugation at 9,600 rpm for 30 min at 4⁰C, 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, CA).

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 X 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. 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. 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

Column^® (Pierce Biotechnology, Inc, Rockford, II); 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, NC) 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 x g for 2 minutes; h. the activated antibody and activated thio-DNA was then mixed and the mixture incuDateα in tne dam at room temperature for 1 hour then overnight at 4° C; i. product from step h was analyzed by gel electrophoresis.

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.

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.

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 50μg 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 50μg 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.

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

REFERENCES

1. Brown, G. W., D. M. Robinson, D. L. Huxsoll, T. S. Ng, K. J. Lim, and G. Sannasey. 1976. Scrub typhus: a common cause of illness in indigenous populations. Trans. R. Soc. Trop. Med. Hyg. 70:444-448.

2. Brown, G. W., J. P. Saunders, S. Singh, D. L. Huxsoll, and A. Shirai. 1978. Single dose doxycycline therapy for scrub typhus. Trans. R. Soc. Trop. Med. Hyg. 72:412-416. 3. Watt G, C. Chouriyagune, R. Ruangweerayud, P. Watcharapichat, D. Phulsuksombati, K. Jongsakul, P. Teja-Isavadham, K. Bhodhidatta, K.D. Corcoran, G.A. Dasch, D. Stickman. 1996. Scrub typhus poorly responsive to antibiotics in northern Thailand. Lancet 348:86-89.

4. Watt G, P. Kantipong, K. Jongsakul, P. Watcharapichat, D. Phulsuksombati, D. Strickman. 2000. Doxycycline and rifampicin for mild scrub-typhus infections in northern Thailand: a randomised trial. Lancet 356: 1057-1061.

5. Mathai E, J.M. Rolain, G.M. Verghese, O.C. Abraham, D. Mathai, M. Mathai, D. Raoult. 2000. Out break of scrub typhus in southern India during the cooler months. Ann. New York Acadamy 990: 359-364.

6. Smadel, J.E., H.L. Ley, Jr., F.H. Diercks, R. Traub, VJ. Tipton, and L.P. Frick. 1951. Immunization against scrub typhus. I. Combined living vaccine and chemoprophylaxis in volunteers. Am.J.Hyg. 53:317-325.

7. Smadel, J.E., H.L. Ley, Jr., F.H. Diercks, P. Y. Paterson, CL. Wisseman, Jr., and R. Traub. 1952. Immunization against scrub typhus: duration of immunity in volunteers following combined living vaccine and chemoprophylaxis. Am.J.Trop.Med.Hyg. 1 :87-99. 8. Eisenberg, G.H., Jr., and J.V. Osterman. 1979. Gamma-irradiated scrub typhus immunogens: broad-spectrum immunity with combinations of rickettsial strains. Infect. Immun. 26:131-136.

9. Eisenberg, G. H., Jr., and J.V. Osterman. 1978 Effects of temperature on the stability of Rickettsia tsutsugamushi and Gamma-irradiated scrub typhus immunogens. Infect. Immun. 22:298-300

10. Ohashi, N., A. Tamura, H. Sakurai, and T. Suto. 1988. Immunoblotting analysis of anti- rickettsial antibodies produced in patients of tsutsugamushi disease. Microbiol. Immunol. 32: 1085-1092

11. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Feigner PL. 1990. uirect gene iransier into mouse

in vivo. Science 247:1465-68.

12. Hoffman, S.L., D.L. Doolan, M. Sedegah, J.C. Aguiar, R. Wang, A. Malik, R.A. Gamizinski, W.R. Weiss, P. Hobart, J.A. Norman, M. Margalith and R.C. Hedstrom. 1997. Strategy for development of a pre-erythrocytic Plasmodium falciparum DNA vaccine for human use. Vaccine (15): 842-845.

13. Coker, C. M. Majid and S. Radulovic. 2003. Development of Rickettsia prowazekii DNA vaccine. Annals of N.Y. Acad, of Sci. 990:757-764.

14. Kelly, D. J., G. A. Dasch, T. C. Chye, and T. M. Ho. 1994. Detection and characterization of Rickettsia tsutsugamushi (Rickettsiales: Rickettsiaceae) in infected Leptotrombidium {Leptotrombidium) fleteheri chiggers (Acari: Trombiculidae) with the polymerase chain reaction. J. Med. Entomol. 31 :691-699.

15. Kim, I.J., I.S. Kim, I.H. Choi, W.H. Chang and M.S. Choi. 1994. Characterization of gene for 47 kDa protein of Rickettsia tsutsugamushi. GENEBANK Accession AAA26374 (19-04- 1994). 16. Walsh, K.A., and H. Neurath. 1964. Tripsinogen and chymotrypsinogen as homologous proteins. Proc. Natl. Acad. Sci. (USA) 52:884.

17. Zumbrunn, J., and B. Trueb. 1996. Primary structure of a putative serine protease specific for IGF-binding proeins. FEBS Lett. 398: 187-192.

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.

Claims

We claim:

1. An immunogenic composition comprising: c. a plasmid containing the DNA sequence encoding the entire or a fragment of the O. tsutsugamushi 1 10 kDa protein, wherein said protein is expressed; and d. a plasmid containing the DNA sequence encoding a cytokine adjuvant, wherein said cytokine adjuvant is expressed and wherein an immune response is induced.

2. The immunogenic composition of claim 1, wherein said DNA encoding said entire or fragment of O. tsutsugamushi 1 10 kDa protein is derived from the O. tsutsugamushi strain selected from the group consisting of Karp, Kato and Gilliam.

3. The immunogenic composition of claim 1, wherein said plasmid expressing said 1 10 kDa protein or fragment is VR 1020.

4. The immunogenic composition of claim 1, wherein said DNA sequence encoding all or a fragment of the O. tsutsugamushi 110 kDa protein is all or a fragment of DNA sequences selected from the group consisting of SEQ ID No.

5,

6, and 7. 5. The immunogenic composition of claim 1, wherein said DNA sequences encode a polypeptide sequence selected from the group consisting of SEQ ID No. 8, 9 and 10.

7. The immunogenic composition of claim 1, wherein said cytokine adjuvant is selected from the group consisting of IL- 12 and GM-CSF.

7. A method of inducing an immune response comprising the 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 thel 10 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.

8. The method of inducing an immune response of claim 7, wherein said boosting dose comprises 2 -10 mg per dose of a plasmid containing the DNA sequence encoding the entire or fragment 110 kDa protein selected from the group consisting of SEQ ID No. 5, 6 and 7 from one or more O. tsutsugamuchi strains selected from the group consisting of Karp, Kato and Gilliam, wherein said protein is expressed and where said boosting DNA sequence is from the same strain as in the priming dose.

9. The method of inducing an immune response of claim 8, wherein said boosting dose also "includes a plaSmid^"containϊng the DNA sequence encoding a cytokine adjuvant selected from the group consisting of IL-12 and GM-GSF, wherein said cytokine adjuvant is expressed.

10. The method of inducing an immune response of claim 7, wherein said boosting dose is the entire or fragment of one or more 1 10 kDa protein of polypeptide sequences selected from the group consisting of Seq ID No 8, 9 and 10 from one or more O. tsutsugamuchi strains selected from the group consisting of Karp, Kato and Gilliam and where said polypeptide sequences are from the same strain or strains as in the priming dose.

11. The method of inducing an immune response of claim 7, wherein said plasmid containing said 1 10 kDa protein or fragment is VR 1020.

12. The method of inducing an immune response of claim 7, wherein said DNA sequences encode a polypeptide sequence selected from the group consisting of SEQ ID No. 8, 9 and 10.

13. The method of inducing an immune response of claim 7, wherein said DNA sequence encoding all or a fragment of the O. tsutsugamushi 1 10 kDa protein is all or a fragment of DNA sequences selected from the group consisting of SEQ ID No. 5, 6, and 7.

14. The method of claim 7, wherein said cytokine adjuvant is selected from the group consisting of IL-12 and GM-CSF.

15. A diagnostic antigen comprising a recombinant antigen of O. tsutsugamushi 110 kDa antigen derived from the O. tsutsugamushi strains selected from the group consisting of Karp, Kato and Gilliam.

16. The diagnostic antigen of claim 15, wherein said recombinant antigen is encoded by all or a fragment of the DNA sequences of DNA sequences selected from the group consisting of SEQ ID No. 5, 6 and 7.

17. The diagnostic antigen of claim 15, wherein said recombinant antigen is inserted in the plasmid pET24.

18. The diagnositic antigen of claim 15, wherein said recombinant antigen is the entire or a fragment of a polypeptide sequence selected from the group consisting of SEQ ID No. 8, 9 and 10.

19. A method of diagnosis of scrub typhus comprising the steps: a. coating wells with one or more than one of the entire or fragment of recombinant polypeptides selected from the group consisting of SEQ ID No. 8, 9 and 10; b. washing said plates to remove excess said polypeptide; c. blocking said plates; d. washing said plates to remove excess said blocking buffer; e. diluting patient sera with said blocking buffer; f. adding said diluted patient sera to said plates; g. incubating said plates containing said sera; h. washing said incubated plates to remove unbound antibody in said patient sera; i. adding to said patient antibody bound plates secondary antibody probe; j. incubating said plates containing added secondary antibody; k. washing said incubated secondary antibody added plates to remove excess secondary antibody;

1. measuring binding of said secondary antibody.

20. The method of claim 19 wherein sensitivity is enhanced by rolling circle amplification wherein said secondary antibody is conjugated with a DNA primer and said DNA primer conjugated secondary antibody is added to said patient antibody bound plates with a mixture containing circular DNA, T7 Sequenase and dATP, dCTP, dGTP and dTTP and fluorescein conjugated dUTP in a reaction buffer containing Tris-acetate, magnesium acetate, potassium acetate and dithiothreotal and conducting said measurment of said binding of secondary antibody by measuring fluorescene incorporation of DNA product.

21. The method of claim 19, wherein sensitivity is enhanced by polymerase chain reaction wherein said secondary antibody is conjugated with a DNA template and said DNA template conjugated secondary antibody is added to said patient antibody bound plates with a reaction mixture containing forward and reverse primers, dATP, dCTP dGTP and dTTP and taq polymerase in a reaction buffer containing Tris-HCl, MgCl₂, and KCl and said incubation of said plates containing said secondary antibody is by alternating cycles of temperature to permit amplification of said template DNA and conducting said measurement of said binding of said secondary antibody by measuring amplified DNA template product.

22. A method of inducing an immune response comprising the steps: a. administering a priming dose comprising 50μg 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 50μg 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.

23. The method of inducing an immune response as in claim 22, wherein said priming dose includes a cytokine adjuvant is added wherein said cytokine adjuvant is selected from the group consisting of IL- 12 and GM-CSF.

24. The method of inducing an immune response as in claim 22, wherein said boosting dose includes said cytokine adjuvant selected from the group consisting of IL- 12 and GM-CSF.