Patent Application
20050234005
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.
This invention relates to the protection against infection of Orientia tsutsugamushi.
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 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].
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.
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 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.
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.
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.
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.
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.
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.
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.
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 MgCl2, 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.
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.
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
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:
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:
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.
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×LD50 of Karp in 0.2 ml of Snyder'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.
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).
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.
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.
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.
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.
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.).
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:
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:
RCA reactions were undertaken the method comprising the following steps:
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:
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.
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.
This application claims priority to U.S. Provisional application 60/563,447 filed Apr. 20, 2004.
| Number | Date | Country | |
|---|---|---|---|
| 60563447 | Apr 2004 | US |
