WO1999010005A1 - Vaccines for chlamydia psittaci infections - Google Patents
Vaccines for chlamydia psittaci infections Download PDFInfo
- Publication number
- WO1999010005A1 WO1999010005A1 PCT/US1998/017943 US9817943W WO9910005A1 WO 1999010005 A1 WO1999010005 A1 WO 1999010005A1 US 9817943 W US9817943 W US 9817943W WO 9910005 A1 WO9910005 A1 WO 9910005A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- momp
- nucleic acid
- polypeptide
- subject
- psittaci
- Prior art date
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Classifications
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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
- C07K14/295—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Chlamydiales (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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
- Y10S530/825—Bacteria
Definitions
- the present invention relates to Chlamydia psittaci vaccines and to methods of protecting animals, including avian species, from Chlamyida psittaci infections.
- Chlamydia contains four species of obligate parasitic bacteria: Chlamydia psittaci, Chlamydia pecorum, Chlamydia pneumoniae, and Chlamydia trachomatis. This unique genus causes a variety of diseases in humans, mammals, and birds. In humans, the most notable are trachoma and urogenital infections due to C trachomatis and psittacosis caused by C. psittaci. In animals, C. psittaci can cause a diverse range of disease in livestock, poultry, turkeys and companion birds. The known C.
- psittaci strains have been grouped into eight biovars (Perez-Martinez, JA and J Storz, 1985). Strains of serovar 1 are mainly associated with intestinal infections and abortions, while strains of serovar 2 cause polyarthritis, encephalitis, and conjunctivitis in ruminants. Avian strains of C. psittaci cause respiratory problems and diarrhea in birds (Storz, 1988). The organism can also be transmitted to humans from these animals, and outbreaks have been documented in animal production workers. Thus, there is a need for an effective vaccine against C. psittaci for mammalian and avian species.
- the chlamydia organism goes through two developmental stages in its life cycle.
- the extracellular form which is the infectious entity of the cycle, is called the elementary body (EB).
- EBs attach and enter the host cell, where they re-organize into reticulate bodies (RBs) which divide within membrane-bound host cell compartments by binary fission and then condense into a new generation of infectious EBs.
- RBs reticulate bodies
- the attachment and entry of the EB into the host cell is a receptor-mediated phenomenon (Hodinka et al. 1988), and several chlamydial proteins have been implicated in the EB attachment to host cellular membranes (Baghian and Schnorr, 1992).
- MOMP major outer membrane protein
- surface-exposed epitopes of this protein from C. trachomatis have been shown to block EB attachment onto the host cell (Su and Caldwell, 1991).
- the MOMP genes from some strains of C. psittaci and C. trachomatis have been sequenced (Baehr et al., 1988, Pickett et al. 1988, Yuan et al. 1989, Zhang et al. 1989, Kaltenboeck, et al. 1993). Analyses of these sequences revealed that portions of the structure of this protein are conserved between species.
- VDl regions of "variable domain" interspersed with conserved sequences, and these are referred to as VDl, VD2, VD3, and VD4.
- VDl regions of "variable domain" interspersed with conserved sequences
- VD2 regions of conserved sequences
- VD3 regions of conserved sequences
- the location of these VD regions are identical in the two species (see Zhang et al., 1989).
- a comparison of the genes encoding the MOMP from C. psittaci and C. trachomatis show that, overall, the sequences are approximately 68% identical.
- the present invention provides a vaccine composition which is protective against Chlamydia psittaci infections in animals, including avian species, comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking regions VDl and VD2. Also provided are polypeptides and isolated nucleic acids encoding such polypeptides, as well as methods of preventing C. psittaci infections in animals comprising administering to the subject animal such vaccine compositions.
- MOMP major outer membrane protein
- nucleic acid vectors for the expression of a MOMP polypeptide-MBP fusion protein comprising a nucleic acid encoding MBP and a nucleic acid encoding an immunogenic C. psittaci MOMP polypeptide arranged in tandem such that the MOMP-MBP fusion protein can be expressed.
- a Chlamydia psittaci infection in a subject comprising administering to the subject a nucleic acid vaccine comprising an immunizing amount of a nucleic acid vector comprising a nucleic acid encoding an immunogenic C. psittaci MOMP polypeptide lacking regions VDl and VD2.
- MOMP means the major outer membrane protein from a Chlamydia psittaci strain.
- polypeptide refers to a polymer of amino acids. As used in combination with MOMP, it means a fragment of MOMP.
- Maltose Binding Protein means a maltose binding protein.
- immunogenic amount means an amount of an immunogen, i.e., a MOMP polypeptide, a MOMP polypeptide-MBP fusion protein, or portions thereof, which is sufficient to induce an immune response in a vaccinated animal and which protects the animal against active infection with Chlamydia psittaci upon exposure thereto.
- immunizing amount means an amount of a nucleic acid expression vector sufficient to induce an immune response in a vaccinated animal and which protects the animal against active infection with Chlamydia psittaci upon exposure thereto.
- Vaccine preparations that are efficacious and economical for use in human and non-human animals, including birds, such as chickens, turkeys and companion birds, are provided.
- Polypeptide vaccines are provided.
- the present invention provides a vaccine composition which is protective against Chlamydia psittaci infections in animals, including avian species, comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking variable regions VDl and VD2.
- the vaccine composition can comprise an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide comprising amino acids 183 through 402 of the MOMP protein from C.
- psittaci strains Avian Type C LSUWTCK (a strain isolated from a cockatiel which has the identical MOMP gene sequence to Avian Type C), or strain 6BC (which is identical to the sequence of Mn except for a single amino acid change), or amino acids 164 to 389 of the MOMP protein from C. psittaci strain B577.
- the complete nucleic acid sequences of the MOMP gene from C. psittaci strains Avian Type C, B577, and 6BC are provided herein as SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, respectively, and the corresponding amino acid sequences of the MOMP proteins are provided herein as SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14, respectively.
- the MOMP polypeptide is purified from other proteins sufficiently to induce a specific immune response.
- the MOMP polypeptide comprising the vaccine comprises the amino acid sequence set forth in SEQ ID NO: 1.
- the MOMP polypeptide of the vaccine comprises the amino acid sequence set forth in SEQ ID NO:2.
- the MOMP polypeptide is provided as a component of a fusion protein.
- a vaccine composition comprising a MOMP polypeptide, as described herein, linked to a non- MOMP polypeptide or protein, and a nucleic acid encoding such a fusion protein.
- a MOMP fusion protein can be made with any desired protein as part of the chimera.
- GST glutathione-S-transferase
- MBP Maltose Binding Protein
- a vaccine composition comprises a Maltose Binding Protein-MOMP fusion protein, wherein the MOMP portion of the fusion protein is a C. psittaci MOMP polypeptide.
- a fusion protein can have MBP as the amino terminal protein of the fusion protein and the MOMP polypeptide as the carboxyl terminal portion of the fusion protein.
- a vaccine composition comprising an MBP-MOMP fusion protein which includes the polyamino acid encoded by nucleotides 1606-2661 of the MBP sequence from the E. coli malE gene (available, for example, in the vector pMALTM-c2 from New England Biolabs, Inc., Beverly, MA 01915-5999).
- a vaccine composition comprising MBP-MOMP fusion protein wherein the MOMP polypeptide is a C. psittaci MOMP polypeptide comprising amino acids 183 through 402 of the MOMP protein from either C. psittaci strains Avian Type C, LSUWTCK, or 6BC, or amino acids 164 to 389 of the MOMP protein from C. psittaci strain B577.
- Fusion proteins utilizing MBP sequences are presented in U.S. Patent No. 5,643,758.
- the fusion protein of this invention has several advantageous characteristics. The unexpected discovery that such a MBP-MOMP fusion protein precipitates in inclusion bodies in the bacterial host cells provided an economical method for purifying this immunogen.
- this MBP-MOMP fusion protein can be expressed from a nucleic acid encoding it in relatively large amounts (e.g., 100 milligrams per liter of E.coli) in a manner such that the fusion protein can be very easily purified to a useful extent.
- MBP-protein fusions are purified by passing the cell extract over an affinity column bound with an appropriate ligand for MBP. The added cost of such a preparation step generally makes such proteins uneconomical as vaccines in production animals.
- the MBP-MOMP fusion protein of this invention can be prepared to sufficient purity without the use of such a column, making the economical production of a production or companion animal vaccine possible. This MBP-MOMP fusion protein precipitates as inclusion bodies, and thus traditional purification techniques, such as affinity columns, are not necessary, and the resulting purified fusion protein retains immunogenicity.
- amino acid substitutions can be selected by known parameters to be neutral substitutions (see, e.g., Robinson WE Jr, and Mitchell WM., AIDS 4:S151-S162(1990)).
- the invention also includes those polypeptides having slight variations in amino acid sequences or other properties. Such variations may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants.
- amino acid sequences of the MOMP polypeptide vaccines of this invention can include sequences in which one or more amino acids have been substituted with another amino acid to provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, alter enzymatic activity, or alter interactions with gastric acidity.
- the peptide must retain C. psittaci- protective immunogenicity.
- variable regions VD3 and VD4 can be administered either in the form of a single MOMP polypeptide, for example SEQ ID NO:l or SEQ ID NO: 2, or as multiple MOMP polypeptides, each one encoding one of the variable regions.
- Techniques for producing such MOMP polypeptides are routine in art, given the knowledge of the full DNA sequence of the MOMP genes. For example, one can construct multiple nucleic acid vectors by first enzymatically cleaving the DNA of the MOMP gene at restriction enzyme sites located on either side and between variable regions VD3 and VD4 and then cloning the resulting DNA fragments into appropriate vectors for expression.
- artificial primers can be designed to amplify the desired portions of the MOMP gene with convenient restriction enzyme recognition sites in the primers, to allow rapid and efficient cloning of selected portions of the MOMP gene into expression vectors.
- Selected MOMP polypeptides can be assayed for immunogenicity and specificity.
- various concentrations of a putative immunogenically specific polypeptide are prepared and administered to an animal and the immunological response (e.g., the production of antibodies or cell mediated immunity) of an animal to each concentration is determined.
- the amounts of antigen administered depend on the subject, e.g., a human or a non-human animal, including a bird, the condition of the subject, the size of the subject, etc.
- an animal so inoculated with the antigen can be exposed to the bacterium to test the potential vaccine effect of the specific immunogenic MOMP polypeptide.
- the specificity of a putative immunogenic MOMP polypeptide can be ascertained by testing sera, other fluids or lymphocytes from the inoculated animal for cross-reactivity with other closely related bacteria.
- the present invention provides an isolated nucleic acid comprising a nucleic acid which encodes a C. psittaci MOMP polypeptide lacking VDl and VD2.
- the nucleic acid includes the variable regions VD3 and VD4 (e.g., amino acids 183 to 402 of the C. psittaci Avian Type C, LSUWTCK, or 6BC MOMP protein, or amino acids 162 to 389 of the C. psittaci B577 MOMP protein.
- the amino terminus end of the isolated nucleic acid can be modified from the native sequence to include an ATG (methionine) start codon and a Kozak regulatory sequence, both of which are typically required for translation in eukaryotes.
- the present invention provides an isolated nucleic acid comprising a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:3.
- This nucleic acid encodes roughly the carboxyl-terminal half of the C. psittaci strain Avian Type C MOMP protein and includes VD3 and VD4. Additionally, the amino terminus end of this sequence has been modified from the native sequence to include an ATG (methionine) start codon and a Kozak regulatory sequence.
- Another embodiment of this invention provides an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 4, which ends at the stop codon TAA, and thus does not include the 3' untranslated sequences included in SEQ ID NO:3.
- the present invention provides an isolated nucleic acid comprising a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 5.
- This nucleic acid encodes roughly the carboxyl-terminal half of the C. psittaci strain B577 MOMP protein and includes VD3 and VD4, and additionally has an ATG (methionine) and a Kozak consensus sequence at the amino terminus of this polypeptide.
- Another embodiment of this invention provides an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 6, which ends at the stop codon TAA, and thus does not include the 3' untranslated sequences included in SEQ ID NO:5.
- the present invention also provides a composition which is protective against C. psittaci infections comprising polypeptides expressed in a suitable host by one or more of the isolated nucleic acids of this invention and a pharmaceutically acceptable carrier.
- nucleic acid vector for the expression of a MOMP polypeptide-MBP fusion protein comprising a nucleic acid encoding MBP and a nucleic acid encoding a MOMP polypeptide arranged in tandem such that the MOMP-MBP fusion protein can be expressed.
- the nucleic acid encodes an amino acid sequence for the MOMP polypeptide selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO:2.
- the nucleic acid encoding MBP is located 5' to the nucleic acid encoding MOMP. Persons skilled in the art are knowledgeable in arranging the nucleic acids such that the fusion protein can be expressed, including ensuring that the reading frame for both nucleic acids is the same, and including various control regions, as also further discussed herein.
- the present invention provides a nucleic acid vector for the transient expression of a C. psittaci MOMP polypeptide in a eukaryotic cell comprising a eukaryotic promoter functionally linked to a nucleic acid encoding a C. psittaci MOMP polypeptide lacking regions VDl and VD2.
- the nucleic acid encodes an amino acid sequence selected from the group consisting of: SEQ ED NO: 7 or SEQ ID NO:8.
- the nucleic acid has a sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
- any desired eukaryotic promoter can be utilized; however, preferable promoters are those that are strong promoters in avian or mammalian cells, as are known in the art and further described below. As discussed herein, certain modifications to the nucleic acid vectors can be made.
- nucleic acid vector for the transient expression of a C. psittaci MOMP polypeptide in a eukaryotic cell comprising a cytomegalovirus promoter functionally linked to a nucleic acid encoding a C. psittaci MOMP polypeptide lacking regions VDl and VD2.
- the nucleic acid encodes an amino acid sequence selected from the group consisting of: SEQ ID NO:7 and SEQ ID NO:8.
- compositions comprising a plurality of the nucleic acid vectors for the transient expression of a C. psittaci MOMP polypeptide in a eukaryotic cell comprising a eukaryotic promoter functionally linked to a nucleic acid encoding amino acid sequence comprising a C. psittaci major outer membrane protein (MOMP) polypeptide lacking regions VDl and VD2.
- MOMP major outer membrane protein
- the present invention provides a composition which is protective against a Chlamydia psittaci infection in animals, including avian species, comprising a plurality of nucleic acid expression vectors comprising a eukaryotic promoter functionally linked to a nucleic acid encoding the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8 in a pharmaceutically acceptable carrier.
- the present invention provides a composition which is protective against Chlamydia psittaci infections in animals, including avian species, comprising a plurality of nucleic acid expression vectors comprising a eukaryotic promoter functionally linked to one or more nucleotide sequences selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 in a pharmaceutically acceptable carrier.
- the nucleic acid expression vector comprises a cytomegalovirus promoter functionally linked to a nucleic acid encoding VD3 and VD4 of MOMP, for example having the nucleotide sequence set forth in either SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
- the nucleic acid encoding the MOMP polypeptide can be any nucleic acid that functionally encodes the MOMP polypeptide.
- the nucleic acid can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
- Preferred expression control sequences are strong and/or inducible promoters such as those derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, Rous sarcoma virus, adenovirus, bovine papilloma virus, etc.
- a nucleic acid encoding a selected MOMP polypeptide can readily be determined based upon the genetic code for the amino acid sequence of the selected MOMP polypeptide, and, clearly, many nucleic acids will encode any selected chimeric protein. Modifications to the nucleic acids of the invention are also contemplated, since mutations can thereby be studied for greater protective vaccine effect.
- modifications that can be useful are modifications to the sequences controlling expression of the MOMP polypeptide to make production of the MOMP polypeptide inducible or repressible upon addition to the cells of the appropriate inducer or repressor.
- Such means are standard in the art (see, e.g.,. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
- the nucleic acids can be generated by means standard in the art, such as by recombinant nucleic acid techniques, as exemplified in the examples herein, and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis.
- the expression vectors of the invention can be in a host capable of expressing the MOMP polypeptide immunogen or the MBP-MOMP fusion protein immunogen.
- a host capable of expressing the MOMP polypeptide immunogen or the MBP-MOMP fusion protein immunogen there are numerous E. coli expression vectors known to one of ordinary skill in the art useful for the expression of the antigen.
- Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
- any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
- the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the antigen. Also, the carboxyl-terminal extension of the antigen can be removed using standard oligonucleotide mutagenesis procedures.
- yeast expression systems can be used. There may be several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MFa-1 gene) is routinely used to direct protein secretion from yeast (Brake et al., 1984).
- the leader region of pre- pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence.
- the antigen coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter.
- the antigen coding sequence is followed by a translation termination codon which is followed by transcription termination signals.
- the antigen coding sequences can be fused to a second protein coding sequence, such as Sj26 or ⁇ -galactosidase, used to facilitate purification of the fusion protein by affinity chromatography.
- a second protein coding sequence such as Sj26 or ⁇ -galactosidase
- the insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast.
- mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein.
- Vectors useful for the expression of antigen in mammalian cells are characterized by insertion of the antigen coding sequence between a strong viral promoter and a polyadenylation signal.
- the vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers.
- the antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary cell line using a methotrexate resistance-encoding vector.
- RNA corresponding to the antigen coding sequence can be confirmed by Northern analysis.
- suitable host cell lines capable of secreting intact human proteins include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc.
- Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
- Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc.
- the vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts.
- vectors for the expression of antigen in mammalian cells those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed.
- the vector can include cytomegalovirus (CMV) promoter sequences and a polyadenylation signal available for expression of inserted DNAs in mammalian cells.
- CMV cytomegalovirus
- nucleic acid vectors for transient expression in a eukaryotic cell are suitable for genetic, or "naked nucleic acid", immunization. These can be constructed using any of a variety of eukaryotic promoters, herein also referred to as -acting transcription/ translation regulatory sequences, known in the art. General methods for the construction, production and administration of nucleic acid vaccines are known in the art, e.g. Vogel, FR and N Sarver (1995) Clin. Microbiol. Rev. 8:406-410.
- nucleic acid vectors comprise nucleic acids that functionally encode, i.e. are functionally linked to a nucleic acid encoding a MOMP polypeptide.
- the nucleic acid can include, for example, expression control sequences, such as a cw-acting transcription/translation regulatory sequence (comprising one or more of the following: a promoter, response element(s), an initiator sequence), an enhancer, and information processing sites, such as ribosome binding sites, RNA splice sites, intron elements, polyadenylation sites, and transcriptional terminator sequences, all of which, either alone or in combinations, are capable of directing expression in the target animal.
- expression control sequences such as a cw-acting transcription/translation regulatory sequence (comprising one or more of the following: a promoter, response element(s), an initiator sequence), an enhancer, and information processing sites, such as ribosome binding sites, RNA splice sites, intron elements, polyadenylation
- Preferred expression control sequences are strong and/or inducible c/ ' s-acting transcription/translation regulatory sequences such as those derived from metallothionine genes, actin genes, myosin genes, immunoglobulin genes, cytomegalovirus (CMV), SV40, Rous sarcoma virus, adenovirus, bovine papilloma virus, etc.
- CMV cytomegalovirus
- SV40 Rous sarcoma virus
- adenovirus bovine papilloma virus
- the C. psittaci MOMP-encoding nucleic acid and expression control sequences are constructed in a vector, such as a plasmid of bacterial origin, for administration to the target animal. There are numerous plasmids known to those of ordinary skill in the art useful for the production of nucleic acid vaccine plasmids.
- a specific embodiment employs constructs using the plasmid "pcDNA3.1 + " as the vector (InVitrogen Corporation, Carlsbad, CA).
- the nucleic acid expression vectors that functionally encode a MOMP polypeptide may additionally contain immunostimulatory sequences ("ISS") that stimulate the animals' immune system.
- ISS immunostimulatory sequences
- Other possible additions to the nucleic acid expression vectors include nucleic acid sequences encoding cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin-12 (IL-12).
- GM-CSF granulocyte macrophage colony stimulating factor
- IL-12 interleukin-12
- nucleic acid expression vectors can be constructed in a non- replicating retroviral vector, such as the Moloney murine leukemia virus (N2) backbone described by Irwin, et al. (1994, J. Virology 68:5036-5044).
- N2 Moloney murine leukemia virus
- the present genes were isolated from C. psittaci, however, homologs from any Chlamydia strain infecting a selected species, can readily be obtained by screening a library from that Chlamydia strain, genomic or cDNA, with a probe comprising sequences of the nucleic acids set forth in the sequence listing herein, or fragments thereof, and isolating genes specifically hybridizing with the probe under preferably relatively high stringency hybridization conditions.
- high salt conditions and/or high temperatures of hybridization can be used.
- the stringency of hybridization is typically about 5°C to 20°C below the T m (the melting temperature at which half of the molecules dissociate from its partner) for the given chain length.
- the nucleotide composition of the hybridizing region factors in determining the melting temperature of the hybrid.
- the recommended hybridization temperature is typically about 55-58°C.
- the C. psittaci MOMP sequence can be utilized to devise a probe for a homolog in any specific animal by determining the amino acid sequence for a portion of the C. psittaci protein, and selecting a probe with optimized codon usage to encode the amino acid sequence of the homolog in that particular animal.
- the present invention contemplates cells containing a nucleic acid expression vector of the invention.
- a cell containing a nucleic acid expression vector encoding a MOMP polypeptide typically can replicate the DNA and, further, typically can express the encoded MOMP polypeptide.
- the cell can be a prokaryotic cell, particularly for the purpose of producing quantities of the nucleic acid, or a eukaryotic cell, particularly a mammalian cell.
- the cell is preferably a mammalian cell for the purpose of expressing the encoded protein so that the resultant produced protein has mammalian protein processing modifications.
- the cell also can be a eukaryotic cell in a host organism, for the purposes of vaccinating the host via genetic or "naked nucleic acid" immunization. In the case of genetic immunization, the nucleic acid expression vector does not typically replicate in the host.
- a nucleic acid expression vector of this invention is administered in combination with one or more other nucleic acid vectors, as a "naked nucleic acid immunization" to protect against multiple viral diseases.
- the other viral diseases can be avian polyomavirus, Pacheco's disease virus, or psittacine beak and feather disease virus.
- the vaccine composition comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking variable regions VDl and VD2 is administered in combination with one or more recombinant viral proteins from viruses that infect and cause disease in psittacine birds.
- MOMP major outer membrane protein
- Any vaccine composition of this invention can further comprise an adjuvant suitable for use in the species to which the vaccine is to be administered, such as avian or mammalian species.
- adjuvants include but are not limited to a cytokine, such as a lymphokine, a monokine or a chemokine, or a cytokine inducer or an agent that facilitates the entry of the antigen into the cytoplasm of the cell.
- cytokine such as a lymphokine, a monokine or a chemokine, or a cytokine inducer or an agent that facilitates the entry of the antigen into the cytoplasm of the cell.
- Other examples of adjuvants that can useful in the present invention include but are not limited to plasmid DNA or bacterial agents.
- An adjuvant can also include, for example, immunomodulators and co-stimulatory molecules.
- Additional adjuvants include any compound described in Chapter 7 (pp 141-227) of 'Vaccine Design, The Subunit and Adjuvant Approach' (eds. Powell, M. F. and Newman, M. J.) Pharmaceutical Biotechnology, Volume 6, Plenum Press (New York).
- Any vaccine composition of this invention can further comprise a pharmaceutically acceptable carrier.
- pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
- a pharmaceutically acceptable carrier can comprise saline or other suitable carriers (Arnon, R, (Ed.) Synthetic Vaccines 1:83-92; CRC Press, Inc., Boca Raton, Florida 1987).
- the present invention further provides methods of vaccinating a subject to induce an immunological response capable of preventing a subsequent C. psittaci infection.
- the vaccine can be administered to an animal of the avian species, such as poultry, turkeys and companion birds; additionally, particularly for handlers of such avian species, the vaccines can be administered to mammals such as humans.
- birds which can be treated by the invention can be any of the various species of birds which are classified as being members of the Psittaciformes order.
- Such birds include, but are not limited to, Budgerigars (Melopsittacus undulatus), caiques (e.g., Pionites leucogaster leucogaster), macaws (e.g., Ara araraund), Amazon parrots (e.g., Amazona ochrocephala auropalliata, conures (e.g., Pyrrhara picta, Aratinga wagleri wagleri, Aratinga solstitialis, Aratinga guarouba, Aratinga holochlora rubritorquis or Aratinga acuticaudata acuticaudat ⁇ ), cockatoos (e.g., Cacatua moluccensis, Cacatua ducorps, Cacatua sulphura, Cacatua gof ⁇ ni or Cacatua alba), Splendid Parakeets (Neophema spectaculard), Pionus
- Budgerigars Me
- Parrots (Pionus maximillan ⁇ ), African Grey Parrots (Psittacus erithacus erithacus, Eclectus Parrots (Electus roratus), Cockatiels (Nymphicus hollandicus) and parakeets (e.g. Psittacula krameri krameri).
- the present invention provides a method of preventing a Chlamydia psittaci infection in a subject comprising administering to the subject a vaccine comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking variable regions VDl and VD2.
- a vaccine comprising an immunogenic amount of a C. psittaci major outer membrane protein (MOMP) polypeptide lacking variable regions VDl and VD2.
- the method comprises an immunogenic amount of a MOMP polypeptide having an amino acid sequence as set forth in either SEQ ID NO: 1 or SEQ ID NO:2.
- the method comprises an immunogenic amount of an MBP-MOMP polypeptide fusion protein.
- the present invention further provides a method of preventing a Chlamydia psittaci infection in a subject comprising administering to the subject a vaccine comprising an immunizing amount of a nucleic acid vector for the transient expression in a eukaryotic cell comprising a eukaryotic promoter functionally linked to a nucleic acid encoding a C. psittaci MOMP polypeptide lacking regions VDl and VD2 of MOMP.
- the method comprises a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO:7 or SEQ ID NO:8.
- the present invention provides a method of preventing a Chlamydia psittaci infection in a subject comprising administering to the subject a vaccine comprising an immunizing amount of a nucleic acid vector for transient expression in a eukaryotic cell comprising a cytomegalovirus promoter functionally linked to a nucleic acid having the nucleotide sequence set forth in either SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
- compositions can be administered to a subject or an animal model by any of many standard means for administering the particular composition.
- compositions can be administered orally, sublingually, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, topically, transdermally, or the like.
- Compositions can be administered, for example as a complex with cationic liposomes, encapsulated in anionic liposomes, or encapsulated in microcapsules.
- Compositions can include various amounts of the selected composition in combination with a pharmaceutically acceptable carrier and, in addition, if desired, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
- Parental administration if used, is generally characterized by injection.
- Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
- Actual methods of preparing dosage forms are known, or will be apparent, to those skilled in this art; for example, see Martin, E.W., Ed., Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA.
- a vaccine of this invention is administered on a regular booster schedule, for example, every six months, to companion birds of the order Psittaciformes.
- the vaccine may be advantageously administered to such birds orally, such as in pill form, or intranasally in a spray, or intraocularly in a drop.
- the vaccine may be administered intramuscularly or subcutaneously.
- MOMP gene sequence of C. psittaci strain B577 isolated from an aborted ovine fetus was identical to the MOMP gene sequence from a C. psittaci strain isolated from a parakeet.
- Primer 3GPB (ACGAATTCCTAGGTTCTGATAGCGGGAC; SEQ ID NO: 15) and primer cBamA (CGGATCCATTACCCAAGGTGTTATGGA; SEQ ID NO: 17) were used for PCR amplification of DNA from the cockatiel C. psittaci strain ("LSUWTCK").
- Primer cBamA was specifically designed to create a BamHI restriction site (GGATCC) for subsequent cloning purposes.
- primer ParaBamG (TAAAGGATCCGCCATGGCAGC; SEQ ID NO: 18), which includes a BamHI site naturally present in the B577 sequence, was used in combination with primer 3GPB.
- the underlined bases in primer ParaBamG represent changes from the natural B577 sequence that create a start ATG codon and a Kozak consensus sequence.
- the PCR-derived DNA fragments which contain either the C. psittaci strain
- the fragment from strain LSUWTCK encodes 222 amino acids from the MOMP gene and starts immediately after the second variable epitope (VD2) and includes all downstream regions (see Zhang et al.1989).
- the fragment from strain B577 encodes 228 amino acids from the MOMP gene and also includes regions VD3 and VD4.
- pMALTM-c2 New England Biolabs, Inc., Beverly, MA 01915-5999.
- MBP maltose binding protein
- the BamHI site in the bacterial expression vector pMALTM-c2 is in frame with the MBP gene.
- the BamHI site in the cBamA synthetic oligonucleotide primer was designed to be in the same reading frame with the MOMP gene. Consequently, the construct described herein will result in the expression of a MBP-MOMP fusion protein.
- primer cBamA has a base change from the native sequence, (G - A), creating a start codon (ATG) in frame with the MOMP coding sequence, so that the ribonucleic acid transcribed from the cloned sequence can be translated in eukaryotic cells.
- the particular location for the ATG was chosen due to the ability to create a Kozak box consensus sequence, G(A). 3 NNATGG(A) +4 around the start site, which enhances translatability in eukaryotes.
- Protein production was carried out according to the instructions included in the manual from New England Biolabs, Inc. (Beverly, MA 01915-5599), except that cells were harvested after 3 hours of induction with Isopropyl-thio-galactoside (IPTG) with a final concentration of 0.3 mM.
- IPTG Isopropyl-thio-galactoside
- the pellet of the cells derived from 500 mL of media was resuspended in 25 mL of lysis buffer: 100 mM NaCl, 100 mM Tris HC1 pH 8.0, 100 mM EDTA pH 8.0, and 10 mM EGTA pH 8.0.
- Fresh lysozyme 10 mg/mL (0.5 mL for 500 mL of media) was added and the sample was left on ice for 30 min at which time 1 mL of 25% Triton XI 00 (T100) was added and left on ice for 5 min. At this time the solution became viscous due to the released DNA.
- the resultant solution after cell lysis was sonicated (5 times, 30 sec. each time or until the viscosity was eliminated) and subsequently 50 mL of lysis buffer was added.
- the sample was centrifuged at 5000 g, 20 min. at +4 °C.
- the supernatant of the sample was discarded and the insoluble part containing the MBP-MOMP fusion protein in the form of inclusion bodies was recovered in the pelleted material.
- the pellet was washed 2 times with lysis buffer + 0.5% T 100 (49 mL of lysis buffer plus 1 mL of 25% T100) using a 10 min incubation time at room temperature.
- the recovered material was resuspended in 25 mL of Lysis buffer without T100 and stored overnight on ice.
- the material was centrifuged at 5000 g, 20 min, +4°C, resuspended in 5 mL of Lysis buffer, added 45 mL of 6M urea subsequently incubated for 15 min at room temperature, and finally centrifuged at 5000 g, 20 min at +4°C.
- the supernatant was recovered and diluted 1 :4 with 5M urea and filtered through an AMICON DIAFLO® Ultrafilter, series XM (Amicon, Lexington, MA 02173), which had a molecular weight cutoff at 50,000, until the volume was approximately 10 mL.
- the total volume was adjusted with 5 M urea to 50 mL and the sample was dialyzed against Phosphate buffer saline (PBS) to decrease the urea concentration in the sample.
- PBS Phosphate buffer s
- the first dialysis step was 2 hours against 100 mL of 2.5 M of urea, and the next 5 steps were against two-fold serially decreasing concentrations of 2.5 M urea.
- Four additional changes of dialysis buffer were performed using 5000 mL of PBS without urea.
- the final product was used for injections.
- the approximate yield of protein extracted from the cells grown in 1 Liter of media was 100 mg, resulting in a MOMP-MBP fusion protein purity of approximately 85%.
- a pool of monoclonal antibodies raised against the C. psittaci strain 6BC and the C. pecorum MOMPs which reacted with purified, native MOMP proteins from strains B577 and 6BC, also reacted with the MBP-MOMP fusion protein in western assays, demonstrating that the fusion protein retains epitopes found on the native protein.
- A. Immunoblotting Assay Cockatiel C. psittaci strain LSUWTCK, grown in Vero cells, was partially purified (Baghian, et al., 1990), resolved in SDS-PAGE and transferred onto nitrocellulose membranes (NCM). Strips cut from the blot were used to evaluate the antibody response to MOMP in vaccinated birds. A rabbit anti-cockatiel IgG produced in the inventors' laboratory was used to detect the cockatiel IgG responses to vaccines. Strips were blocked, reacted with cockatiel serum, then with the rabbit anti-cockatiel IgG followed by exposure to a hydrogen peroxidase conjugated goat anti-rabbit IgG and color-forming substrate.
- Groups II, IH, and IV were vaccinated with the vaccines of this invention.
- Group V the birds were inoculated with inactivated elementary bodies from C. psittaci.
- the Groups were set up as follows:
- Group ⁇ pcDN A 3.1 ZeoVCpMOMP DNA vaccine Group HI: MBP-MOMP fusion protein vaccine (Example 3 A) Group IV: pcDNA 3.1 Zeo7C ⁇ MOMP DNA and MBP-MOMP fusion protein combination vaccine Group V: Vaccination with inactivated EBs
- Group H pcDNA 3.1 ZeoVCpMOMP DNA vaccine
- Group rV Vaccination with inactivated (by irradiation) EBs
- Group V Vaccination with glutaraldehyde-treated, inactivated EBs
- Group I did not receive any vaccinations.
- Group II 100 microliters of pcDNA 3.1 ZeoVCpMOMP DNA (1 microgram/microliter) mixed with 100 microliters of PBS. This mixture was injected in 4 sites using 50 microliters/site.
- 1.7 milliliters (mL) of the MOMP-MBP fusion protein (1 microgram/microliter) was mixed with 1.7 mL Adju-Phos [Aluminum Phosphate Gel adjuvant (Superfos Biosector a/5, Inc., Denmark)], and 0.4 microliters were injected subcutaneously in each bird.
- Adju-Phos Adju-Phos [Aluminum Phosphate Gel adjuvant (Superfos Biosector a/5, Inc., Denmark)]
- 0.4 microliters were injected subcutaneously in each bird.
- each bird simultaneously received the same inoculations of Group II and Group HI, therefore a combination "fusion-protein/DNA" vaccine.
- Group V
- Group I did not receive any vaccinations.
- Group II 100 microliters of pcDNA
- ZeoVCpMOMP DNA (1 microgram/microliter) mixed with 100 microliters of PBS. This mixture was injected as follows: 100 microliters were dropped into the nasal canals, 50 microliters into each side of the nose, and 100 microliters was injected at 3 sites in the chest muscle.
- 1.7 milliliters (mL) of the MOMP-MBP fusion protein (1 microgram/microliter) was mixed with 1.7 mL Adju-Phos [Aluminum Phosphate Gel adjuvant (Superfos Biosector a/5, Inc., Denmark)], and 0.4 microliters were injected subcutaneously—injections were performed as described for Group II.
- the birds were injected with inactivated elementary bodies from C. psittaci, inactivated either with cobalt source irradiation, or made according to the protocol in C.3. below, and then further treated by irradiation with a cobalt source.
- Group II intramuscularly
- Group HI subcutaneously
- Group IV as in Group ⁇ and HI
- Group V subcutaneously On November 4, 1996, the four groups were given second injections, identical to the ones indicated above. On December 19, 1996, the four groups were given third injections, identical to the 1st and 2nd injections. On January 14, 1997, the birds were bled again, then challenged with a 0.5mL suspension of C. psittaci EBs containing at least 10 4 , preferably 5 x 10 5 , infection forming units (IFU). These challenges were administered intranasally and by ocular drops, one in each eye.
- IFU infection forming units
- Y_ Experiment B-l Seroconversion Bird sera taken after the third vaccine inoculation, just before the challenge with infectious EBs, were used in a immunoblotting assay. Among the four groups which were vaccinated, only Group HI showed consistent seroconversion, in that two birds were strongly positive while three birds were weakly positive. One of the birds in Group V which was vaccinated with inactivated EBs also gave a strong reaction in the immunoblotting assay.
- Group I This group of birds exhibited signs of bilateral conjunctivitis, irritated choana and stained vents (a sign of diarrhea). One bird in the group died 38 days post-exposure, and C. psittaci was cultured from this animal. Most birds in this group showed clinical improvement after 25 days post-exposure, until they were sacrificed. Group H. The majority of this group showed mild, clinical signs associated with conjunctivitis, and an irritated choana (upper respiratory irritation). Little or no gastrointestinal abnormalities were noted. All birds survived and were sacrificed.
- Group IH Three birds in this group showed very few clinical abnormalities during the trial, indicating that they were protected from the challenge. On days 3-5 after the challenge, bilateral conjunctivitis, irritated choanas and vent staining was apparent. Two of the birds died during the trial period.
- Group IV Four birds from this group exhibited minimal conjunctivitis through the trial period. One bird did show bilateral conjunctivitis with severe diarrhea, and it died early in the trial period.
- Group V The entire group of birds had bilateral conjunctivitis and choanal irritation through 75% of the vaccine trial period. This group of birds survived with minimal upper respiratory clinical abnormalities. No gastrointestinal abnormalities were noted. All birds were sacrificed at the end of the trial period.
- Group I birds showed moderate clinical signs of Chlamydia psittaci infection (see the description under Group I in Experiment B-l above).
- Groups H and HI were rated as being normal, i.e. all birds were clinically normal, except for a rare abnormality noted, such as conjunctivitis or nasal discharge, which were not considered to be treatment-related.
- vaccines may be delivered to the animals at the sites of potential invasion by C. psittaci, e.g. the oral or nasal mucosa.
- birds may be vaccinated by an intranasal route.
- Preparations of vaccine, as described in Example 4 can be administered to the nasal mucosa via a spray delivered intranasally to the bird, or through aerosolization of the vaccine. The latter may be effective when a large number of animals is to be vaccinated simultaneously.
- a vaccination protocol appropriate for particular animal(s)' needs (see Tizard, Ian; "Veterinary Immunology: An Introduction", Fourth Edition, 1992, W.B. Saunders Company, Philadelphia, PA, 498 pp.).
- the vaccines of this invention can also be administered through the feed or in the drinking water of the animal.
- Baghian, A and K Schnorr. 1992 Detection and antigenicity of chlamydial proteins which bind eukaryotic cell membrane proteins. Baghian, A, L Shaffer, and J Storz. 1990. Antibody response to epitopes of chlamydial major outer membrane proteins on infectious elementary bodies and of the reduced polyacrylamide gel electrophoresis-separated form. Infect. Immun. 58:1379-1383.
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AU90393/98A AU757762B2 (en) | 1997-08-28 | 1998-08-28 | Vaccines for chlamydia psittaci infections |
EP98942307A EP1017409A4 (en) | 1997-08-28 | 1998-08-28 | Vaccines for chlamydia psittaci infections |
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US60/057,147 | 1997-08-28 |
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EP (1) | EP1017409A4 (en) |
AU (1) | AU757762B2 (en) |
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Cited By (4)
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WO2000053764A1 (en) * | 1999-03-12 | 2000-09-14 | Aventis Pasteur Limited | Chlamydia antigens and corresponding dna fragments and uses thereof |
WO2001059456A2 (en) * | 2000-02-11 | 2001-08-16 | Yaba Limited | Compounds for detection of infection by chlamydophila abortus |
US6899880B2 (en) | 2000-02-01 | 2005-05-31 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by chlamydia |
US7105171B2 (en) | 2002-03-07 | 2006-09-12 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia |
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ATE318924T1 (en) * | 1998-12-04 | 2006-03-15 | Univ Manitoba | TWO-STEP PROCESS FOR VACCINATION AGAINST CHLAMYDIA |
AU784193B2 (en) * | 1999-12-22 | 2006-02-16 | Sanofi Pasteur Limited | Chlamydia antigens and corresponding DNA fragments and uses thereof |
PL2729158T3 (en) * | 2011-06-17 | 2018-07-31 | Universiteit Gent | Vaccines for chlamydia |
WO2016081327A2 (en) * | 2014-11-21 | 2016-05-26 | Merck Sharp & Dohme Corp. | Recombinant expression of chlamydia momp antigen |
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US5725863A (en) | 1991-09-06 | 1998-03-10 | The United States Of America As Represented By The Secretary Of Agriculture | Polypeptides useful in prevention of chlamydia infection |
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- 1998-08-28 US US09/143,127 patent/US6605287B2/en not_active Expired - Lifetime
- 1998-08-28 AU AU90393/98A patent/AU757762B2/en not_active Expired
- 1998-08-28 EP EP98942307A patent/EP1017409A4/en not_active Withdrawn
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2003
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WO2000053764A1 (en) * | 1999-03-12 | 2000-09-14 | Aventis Pasteur Limited | Chlamydia antigens and corresponding dna fragments and uses thereof |
US6899880B2 (en) | 2000-02-01 | 2005-05-31 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by chlamydia |
US8105607B2 (en) | 2000-02-01 | 2012-01-31 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia |
WO2001059456A2 (en) * | 2000-02-11 | 2001-08-16 | Yaba Limited | Compounds for detection of infection by chlamydophila abortus |
WO2001059456A3 (en) * | 2000-02-11 | 2001-12-27 | Yaba Ltd | Compounds for detection of infection by chlamydophila abortus |
US7105171B2 (en) | 2002-03-07 | 2006-09-12 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia |
US7253275B2 (en) | 2002-03-07 | 2007-08-07 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia |
US7670855B2 (en) | 2002-03-07 | 2010-03-02 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia |
US8025891B2 (en) | 2002-03-07 | 2011-09-27 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia |
US8337866B2 (en) | 2002-03-07 | 2012-12-25 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by chlamydia |
US8679510B2 (en) | 2002-03-07 | 2014-03-25 | The Regents Of The University Of California | Porin B (PorB) as a therapeutic target for prevention and treatment of infection by Chlamydia |
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US7279171B2 (en) | 2007-10-09 |
US6605287B2 (en) | 2003-08-12 |
US20020136742A1 (en) | 2002-09-26 |
AU757762B2 (en) | 2003-03-06 |
EP1017409A4 (en) | 2001-05-16 |
AU9039398A (en) | 1999-03-16 |
EP1017409A1 (en) | 2000-07-12 |
US20050037019A1 (en) | 2005-02-17 |
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