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WO2006078271A2 - Clonage et expression de l'antigene complet 110 kda de orientia tsutsugamushi pouvant servir de composant vaccinal contre le typhus des broussailles - Google Patents

Clonage et expression de l'antigene complet 110 kda de orientia tsutsugamushi pouvant servir de composant vaccinal contre le typhus des broussailles Download PDF

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WO2006078271A2
WO2006078271A2 PCT/US2005/013255 US2005013255W WO2006078271A2 WO 2006078271 A2 WO2006078271 A2 WO 2006078271A2 US 2005013255 W US2005013255 W US 2005013255W WO 2006078271 A2 WO2006078271 A2 WO 2006078271A2
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dna
fragment
seq
immune response
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WO2006078271A3 (fr
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Wei-Mei Ching
Chien-Chung Chao
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US Department of Navy
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/29—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Richettsiales (O)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0233—Rickettsiales, e.g. Anaplasma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A61K2039/55527—Interleukins
    • A61K2039/55538—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • 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
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 0 C, 4 min at 94°C, and followed by 30 cycles of 94 0 C for 1 min, 57 0 C for 2 min and 72 0 C for 2 min. The last cycle was extended for 7 min at 72 0 C.
  • 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.
  • VR 1020 was utilized, any plasmid or viral expression system can be used as long as polypeptide is expressed.
  • 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).
  • 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 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 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 ⁇ ' TO ' D ' NA as a vaccine candidate in mouse model.
  • mice 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 LD 50 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.
  • pKp 1 10 DNA against challenge in mouse model is summarized in Table 1 and Table 2.
  • Table 1 The protective efficacy of pKp 1 10 DNA against challenge in mouse model is summarized in Table 1 and Table 2.
  • 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.
  • 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.
  • 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).
  • 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
  • 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.
  • 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).
  • 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 ⁇ g/ml kanamycin at 37 0 C to an O.D. ⁇ oo 0.8 at which time IPTG (1 mM) was added. The culture was then incubated with shaking for 4 hrs at 37 0 C. Cells were harvested and weighted (about 5.5g of wet cells per liter culture')". The cell " pe ⁇ efs were re-suspended in 7 volume of buffer A (20 mM Tris-HCl, pH
  • 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.
  • 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 TM , Agilent Technology, Palo Alto, CA).
  • O. tsutsugamushi peptides can be utilized either alone or in combinantion with other O. tsutsugamushi fragment antigens in immunodiagnostic assays comprising the following steps:
  • Microtiter plates with 96 wells were coated with 0.3 ⁇ g/well of any or all of the recombinant proteins represented by SEQ ID No. 17 - 22 and stored in 4° C for 2 days.
  • 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).
  • RCA antibody specific to O. tsutsugamuchi is conjugated with a single stranded DNA primer comprising the following steps:
  • 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 incuDate ⁇ in tne dam at room temperature for 1 hour then overnight at 4° C; i. product from step h was analyzed by gel electrophoresis.
  • 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.
  • 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 ⁇ g 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 ⁇ g 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.
  • 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.

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Abstract

La présente invention se rapporte à une protéine 110 kDa recombinée de O. tsutsugamuchi , des souches Karp, Kato et Gilliam, et à un système d'expression d'ADN contenant un ADN qui code pour cette protéine 110 kDa de O. tsutsugamuchi. . L'invention concerne également l'utilisation de ces produits de recombinaison dans une préparation destinée à déclencher une réponse immunitaire protectrice contre l'infection par O. tsutsugamuchi . L'invention concerne également une protéine O. tsutsugamuchi 110 kDa ou des fragments 110 kDa permettant la production d'antigènes destinés à des tests d'immunodiagnostic du typhus des broussailles.
PCT/US2005/013255 2004-04-20 2005-04-19 Clonage et expression de l'antigene complet 110 kda de orientia tsutsugamushi pouvant servir de composant vaccinal contre le typhus des broussailles Ceased WO2006078271A2 (fr)

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US60/563,477 2004-04-20

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US7335477B2 (en) * 1997-12-24 2008-02-26 The United States Of America As Represented By The Secretary Of The Navy Truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi strains Karp, Kato and Gilliam and its use in antibody based detection assays and vaccines
ATE493495T1 (de) * 2000-09-08 2011-01-15 Univ Maryland Biotech Inst Genmanipulierte dna-vakzine zur co-expression, methoden zu deren herstellung und deren verwendungen

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