WO2004005535A2 - Pathogenicity proteins which can be used as targets for developing means for preventing and controlling bacterial infections - Google Patents
Pathogenicity proteins which can be used as targets for developing means for preventing and controlling bacterial infections Download PDFInfo
- Publication number
- WO2004005535A2 WO2004005535A2 PCT/EP2003/008209 EP0308209W WO2004005535A2 WO 2004005535 A2 WO2004005535 A2 WO 2004005535A2 EP 0308209 W EP0308209 W EP 0308209W WO 2004005535 A2 WO2004005535 A2 WO 2004005535A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- virulence
- pathogenicity
- serum
- nucleic acids
- targets
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
Definitions
- Pat ogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination
- the invention relates to pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic disseminatio .
- the invention provides a novel strategy, the aim of which is to specifically target pathogenic ba ⁇ teria without significantly altering their growth at their portal of entry into the host organism, where they are in a situation of commensalism.
- pathogens are in particular the bacteria responsible for serious systemic infections, such as E . coli , in general Enterobacteria, Pseudomonas, Acinetobacter,
- Moraxella and Neisseria and, for the gram positives, the bacteria of the genus Staphylococcus , Enter ⁇ COCCUS and Streptococcus .
- the ability of bacteria to grow in human serum is due to different pathogenicity/virulence factors.
- the physical barrier represented by the capsule, for access of complement to the bacterial membrane, the sialic acids of the capsule or of the 0 antigen which promote binding of factor H to c3b, and particular surface proteins such as PorA (Neisseria gonorrhoeae) , YadA ⁇ Yersinia pestis ) or protein M
- Streptococcus pyogenes which bind factor H, all these factors preventing complement activation.
- the lipopolysaccharide (LPS) of gram-negative bacteria is known to be a virulence factor, but the role of its various constituents on the resistance to serum has not been established for all bacterial species.
- the 0 antigen is considered to be determinant (Burns S.M. Hull S.I. Infect I mun, 1998, Sept 66 (9) : 4244-53) ; in other studies, the 0 antigen is thought to be less determinant than the capsular antigens for resistance to serum (Russo T. et al., Infect Immun, 1995, Apr. 63 (4) : 1263-9) .
- the importance of these factors on intestinal colonization is unknown.
- the inventors have carried out a systematic analysis of mutants for inactivation of the genes required for surface polysaccharide synthesis, and have demonstrated, in Escherichia coli strains responsible for extra-intestinal infections, EXPEC, which genes are essential for the resistance to serum and the dissemination in the blood. These results are based on the study of the effect of mutations on virulence and intestinal colonization in an animal model.
- the invention is therefore directed towards a novel methodology for defining the targets required for virulence, and not essential in vi tro, and thus providing novel anti- infectious agents specific for pathogenic bacteria, in particular for extra-intestinal E. coli, responsible for severe infections, as well as Gram positive strains, such as Streptococcus agalactiae . It is also directed towards the products of the genes required for resistance in the serum and virulence in vivo .
- the pathogenic microorganism is in particular an EXPEC strain of E. coli or a Streptococcus agalactiae strain.
- the virulence gene inactivation mutants used in this method fall within the scope of the invention.
- Said mutants are characterized by the following properties : they are sensitive to serum; they are avirulent in mice model and they are able to colonize gut of axenic mice.
- the invention is also directed towards the pathogenicity or virulence factors encoded by nucleic acids thus identified, which are necessary for the dissemination via the blood, but do not significantly affect the intestinal or mucosal colonization of pathogenic bacteria such as E. coli , Salmonella typhimurium, Klebsiella pneumoniae, Yersinia pestis, Serratia arcescens , Haemophilus influenzae, Pasteurella multocida , Vibrio cholerae , Pseudomonas aeruginosa , Acetinobacter , Moraxella catarrhalis , Burkholderia pseudomallei , Neisseria meningitidis , Neisseria gonorrhoeae , Campylobacter jejuni , Helicobacter pylori , Bacteroides fragilis , Clostridium acetobutylicum, Mycobacterium tuberculosis , Streptococcus py
- the invention is in particular directed towards the pathogenicity or virulence targets encoded by isolated or purified nucleic acids having sequences SEQ ID Nos 16-30.
- the pathogenicity or virulence targets of the invention are more particularly encoded by nucleic acids having sequences SEQ ID Nos 16, 17,19-30.
- Said nucleic acids are cDNAs or RNAs. It particularly relates to pathogenicity or virulence targets encoded by nucleic acids of E. coli .
- the pathogenicity or virulence targets are encoded by nucleic acids of Streptococcus agalactiae.
- the invention is also directed towards the vectors comprising at least a nucleic acid coding for a pathologenicity or virulence target such as above defined and also the host cells containing at least one vector under the control of a suitable promoter.
- the invention is also directed towards pathogenicity or virulence factors corresponding to isolated or purified polypeptides or peptides having one of the amino acid sequences SEQ ID Nos 1-15.
- the antibodies which are ' capable of binding specifically to the peptides and polypeptides corresponding to said factors are also part of the invention.
- nucleic acids and peptides or polypeptides constitute targets for identifying compounds with a specific inhibitory effect on the systemic dissemination of a bacterial infection, and not on mucosal colonization or, for enterobacteria, on intestinal colonization, which makes it possible to preserve the commensal flora and to avoid the selection of resistance to the compounds .
- the invention is thus directed towards the method for inhibiting the proliferation of a pathogenic microorganism in serum, comprising the use of an effective amount of a compound capable of inhibiting the activity, or of reducing the amount, of a nucleic acid as defined above, or of a compound capable of inhibiting the activity of a polypeptide or of a peptide as defined above.
- the compounds thus selected are used, in accordance with the invention, to produce medicinal products for inhibiting a bacterial infection, in particular an extra-intestinal infection in the case of enterobacteria .
- the invention thus provides a novel strategy and novel means for preventing or treating systemic bacterial dissemination, bacteraemia and septicaemia.
- Example 1 gene corresponding to SEQ ID N°23:
- the general strategy based on a recombination system, consists in interrupting a gene, by allelic recombination, with a gene for selection (a gene for resistance to antibiotic in the present case) carried by a linear DNA fragment.
- a plasmid is introduced into the bacterium (for example E. coli ) , so as to introduce, in trans, the proteins which will induce the recombination.
- the plasmid carrying an ampicillin resistance gene is thermosensitive (30°C), which will make it possible to easily eliminate it after use in the bacterium.
- the plasmid is introduced into the bacterium by electroporation. After electroporation, the ampicillin- resistant bacteria will be those which have integrated the plasmid, and will be selected. This step is entirely carried out at 30°C, the permissive temperature for the plasmid.
- a PCR is carried out, on a matrix plasmid carrying the selection gene (chloramphenicol resistance) , using primers pg23Pl and pg23P2 of sequences SEQ ID No 31 and SEQ ID No 32, respectively, made up of two parts: in 3' : 20 bp homologous to the selection gene (chloramphenicol resistance) : PI or P2 in 5' : 40 bp homologous to the target gene (pg23) : HI or H2 pg23Pl: 5'
- a DNA fragment consisting of the selection gene (CAT: Chloramphenicol Acetyl Transferase) flanked by the regions homologous to the target gene Hi and H2 is thus obtained.
- the bacterium containing the plasmid is cultured in LB medium at 30 °C with shaking, in the presence of 100 mM ampicillin and of 1 mM L-arabinose so as to induce the recombination system.
- the culture is stopped, and the bacteria are harvested and made electrocompetent .
- the PCR product specific- for the target gene [pg23) is introduced into the bacterium by electroporation.
- the bacteria are then cultured in a non- selective rich medium (SOC medium) at 37 °C with shaking for 2 hours, and then plated out onto selective LB agar medium. After 18 hours at 37 °C, only the bacteria which have integrated the gene for resistance to the antibiotic will have grown.
- SOC medium non- selective rich medium
- PCR reactions are carried out directly using colonies.
- Three pairs of primers are used: a pair in which the primers FR1 and FR2 frame the target gene, and two pairs using a primer located inside the resistance cassette, the other primer being located either upstream or downstream of the target gene.
- the colonies thus verified by PCR are successively re-isolated on selected medium, twice on non-selective medium and a final time on selective medium at 37°C. Finally, the selected bacteria are tested for sensitivity to ampicillin, which reflects the absence of the plasmid. Three clones are thus chosen for each type of mutant.
- the serum used is of human origin.
- growth was also effected for the wild-type strain (S26, clinical isolate of E. coli particularly resistant to serum and virulent in mice) and a strain, ECOR4, lacking a capsule and lipopolysaccharide (LPS) .
- the growths were effected in triplicate and in two different sera.
- the growths were effected in parallel in complemented and decomplemented (30 min at 56°C) serum in order to verify that the effect observed was due only to the lytic action of complement.
- the mutant ⁇ pg23 exhibits considerable sensitivity to the serum: a difference from the wild-type strain of more than 2 log at 1 hour and of more than 4 log at 4 hours is in fact observed.
- the results obtained in decomplemented serum and with the strain ECOR4 in serum indicate that the effect observed is indeed due to the bactericidal action of complement.
- the wild-type mutated bacteria are isolated from the strain, stored at -80 °C, on an LB agar dish with or without antibiotic, and incubated at 37 °C for 18 hours.
- a preculture is prepared in liquid medium. Using a 1/lOth dilution in 10 ml of LB, the culture is regrown at 37 °C with shaking for 2 hours. After culturing for 2 hours, the OD 60 o nm is measured and various dilutions are prepared in physiological saline, so as to obtain the desired inoculum.
- the LD 50 corresponds to an inoculum of 5xl0 5 cfu/mouse and the LD 100 corresponds to an inoculum of 1x10 s cfu/mouse.
- mice (6-week-old Balb/c) are given an intraperitoneal injection and the bacterial solution injected represents a volume of 100 ⁇ l. Five mice are used per dose.
- S26 ⁇ pg23 4 inoculums were tested and the survival rate was measured after 24 and 48 hours post-injection.
- the study was carried out in parallel with the wild-type strain, the LD 5 0 of which is 5xl0 5 cfu/mouse.
- the mutant S26 ⁇ pg23, injected at a dose equal to 10 times the LDioo / causes no mortality, the mutation of the pg23 gene in the E. coli strain Kl S26 is therefore responsible for a considerable decrease in the virulence.
- the entire experiment is carried out in a sterile environment, with sterile instruments, in an isolator, and the mice are given sterile food.
- mice These are 6- to 8-week-old axenic female mice of the C3H/He J line.
- the wild-type and mutated bacteria are isolated from the strain, stored at -80°C, on an LB agar dish with or without antibiotic, and incubated at 37 °C for 18 hours. After culturing the strain in liquid medium, various dilutions are prepared in physiological saline, so as to obtain an inoculum of 10 7 cfu/ml.
- the bacterial inoculation is carried out orally. During the 24 hours preceding inoculation, the mice are deprived of water. They are then given a bacterial solution at IO 7 cfu/ml to drink for 4 hours. The volume of drink is measured at 0 and 4 hours, and, on average, a mouse absorbs 5 ml of this bacterial solution. The faeces are then sampled at various times, and a bacterial count is performed, taking the faeces up in physiological saline and plating out various dilutions on an LB agar dish with and without antibiotic. The results are given in table 1 herein below.
- colonization in the intestine was stably established. No difference is observed between the wild-type strain and the mutant ⁇ pg23. The colonization is confirmed on the final day by removing the intestine and counting the bacteria after grinding of this organ.
- the nucleic acid encoding the polypeptide is cloned into a prokaryotic expression vector such as pET-14b with an N- terminal poly-his tag, according to conventional cloning methods .
- the recombinant plasmid is then used to transform the E . coli strain BL21.
- the transformed cells are selected in the presence of ampicillin and the colonies are isolated. They are then cultured in the presence of IPTG in order to induce expression of the protein.
- the clones producing the protein are cultured and the total proteins are extracted by cell lysis.
- the recombinant protein is purified with a histidine tag affinity column, according to the manufacturer's protocol.
- the protein thus obtained is purified and used in vi tro to measure its enzyme activity.
- Example 2 serum sensitivity and LD 5Q determination of mutant strains in the mice model of infection
- Said mutants were also compared to the wild type S26 E. coli strain for LD 5 o determination in the mice model of infection. As presented in Table 2 below, the number of colony forming unit (cfu) counted after culture for four hours in serum was higher in the wild type (wt) S26 strain than in mutants indicating that mutants were sensitive to serum killing.
- mice lethal dose 50
- mice completely avirulent as no dose killing 50% of mice could be reach with the mutants.
- the bacteria colonizing the intestine of axenic mice after eight days were characterized to verify that they correspond to the mutant strains that were inoculated orally.
- LPS inner core metabolism are not essential in E . coli strains for colonization, but are necessary for resistance to complement and virulence in vivo .
- the present invention relates to novel mutant strain of Group B Streptococcus (GBS) ⁇ Streptococcus agalactiae) .
- the identified targets correspond to gene sequence number 29 encoding a protein sequence number 14 involved in incorporation of D-alanine residues into the cell wall-associated lipoteichoic acids (LTAs) in Gram + bacteria.
- the gene sequence number 29 is homologous to the dl tD gene found in other gram positive bacteria and is the last gene of the dlt operon.
- the Gram + bacterial model used is the pathogenic strain S . agalactiae NEM316.
- S . agalactiae is a bacterium commonly found in the human flora and is phylogeneticaly close to Gram + bacteria responsible for nosocomial septicemia.
- the virulence of GBS mutants in the dlt operon is strongly impaired in mouse and newborn rat models.
- the loss of virulence is presumably due to an increased sensitivity to antimicrobial cationic peptides, such as defensins, which are produced by numerous cells types in particular phagocytes.
- mutant of S . agalactiae in which the dltD gene have been inactivated, demonstrates that the product of that gene is a good target for the development of inhibitors of virulence of S . agalactiae as well as against other Gram + pathogens. Construction of a DtlD mutant in wild type S . agalactiae NEM316:
- a mutant in the dltD gene was constructed from S . agalactiae NEM316 strain by inserting, using double cross-over, a kanamycin resistance cassette.
- DltD mutant of S. agalactiae NEM316 a promoterless and termina torless kanamycin resistance cassette aphA-3 within DNA segment internal to dltD were inserted in the same direction of transcription . This was done by ligation after digestion wi th appropriate enzymes, of PCR products obtained by using the primers of SEQ ID N° 33 and 34 respectively,
- SEQ ID N°33 5 ' -CAGTGAATTCGCGTTGACGAAGGCAGG-3 '
- SEQ ID N°34 5 ' -GACGGGTACCATACCTATCGTAGGTTG-3 '
- primers of SEQ ID N° 35 and SEQ ID N°36 respectively, SEQ ID N°35 : 5 ' -AGTGGATCCACTACACAGGGCTTGATC-3 '
- SEQ ID N°36 5 ' -GACCTGCAGCCCTTGATTATCCCTATCC-3 ' .
- thermosensitive shuttle vector pG+host5 ⁇ aphA-3 (Biswas et al., 1993, J Bacteriol. 175:3628-3635) containing the kanamycin resistance cassette to generate pGl ⁇ EKaphA-3.
- a 0.8 kb closely spaced dltD region BamHI-Pstl fragment was inserted into pGl ⁇ EKaphA-3 to generate pGl ⁇ EKaphA-3BP.
- the resulting vector was introduced by electroporation into NEM316.
- Transformants were selected on Todd-Hewitt (TH) agar plates containing 10 mg l -1 erythromycin at 30 °C. Allelic exchange was obtained at the non-permissive temperature (42°C) by homologous recombination using a two-step procedure described previously (Biswas et al . , 1993).
- the sensitivity of wild type S . agalactiae NEM316 and DltD mutant to cationic antimicrobial peptides was measured by using a disk diffusion methods. The 2 strains were grown on blood agar plates and incubated for 18 hours at 37 °C. Each strain was tested using colistin (50 ⁇ g) and polymixin (10 ⁇ g) disks. Sensitivity . or resistance of NEM316 strain and the DltD mutant to each compound was determined by the size of the growth inhibition area around disk.
- the DltD mutant exhibited an increased sensitivity to the cationic antimicrobial peptides colistin, and polymyxin B as shown in table 5.
- mice Six week-old Balb/c were inoculated intravenously with 5 x IO 7 bacteria. At 2 days post infection,
- FIG. 1 illustrates the results obtained with the DltD defective GBS mutant. The result demonstrates that the product of the dltD gene is necessary for virulence of GBS in mice.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Toxicology (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE60320797T DE60320797D1 (en) | 2002-07-09 | 2003-07-09 | PATHOGENIC DETERMINANTS AS TARGET MOLECULES FOR THE DEVELOPMENT OF AGENTS WHICH CAN PREVENT AND CONTROL BACTERIAL INFECTIONS AND / OR SYSTEMATIC DISSOLUTION |
US10/520,820 US20060003393A1 (en) | 2002-07-09 | 2003-07-09 | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination |
DK03762684T DK1523572T3 (en) | 2002-07-09 | 2003-07-09 | Pathogenicity determinants that can be used as targets for developmental methods for the prevention and control of bacterial infections and / or systemic dissemination |
AU2003266246A AU2003266246A1 (en) | 2002-07-09 | 2003-07-09 | Pathogenicity proteins which can be used as targets for developing means for preventing and controlling bacterial infections |
EP03762684A EP1523572B1 (en) | 2002-07-09 | 2003-07-09 | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination |
JP2004518773A JP2006515155A (en) | 2002-07-09 | 2003-07-09 | Pathogenic determinants that can be used as targets in the development of prevention and control measures for bacterial infection and / or systemic dissemination |
CA002491847A CA2491847A1 (en) | 2002-07-09 | 2003-07-09 | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination |
HK05109367A HK1078902A1 (en) | 2002-07-09 | 2005-10-20 | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination |
US11/506,821 US20070111229A1 (en) | 2002-07-09 | 2006-08-21 | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination |
AU2009201727A AU2009201727A1 (en) | 2002-07-09 | 2009-04-30 | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination |
US12/729,630 US20110236886A1 (en) | 2002-07-09 | 2010-03-23 | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0208636A FR2842210B1 (en) | 2002-07-09 | 2002-07-09 | PATHOGENICITY DETERMINANTS FOR USE AS TARGETS FOR PREPARING AND CONTROLLING BACTERIAL INFECTIONS AND / OR SYSTEMIC DISSEMINATION |
FR0208636 | 2002-07-09 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/506,821 Division US20070111229A1 (en) | 2002-07-09 | 2006-08-21 | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2004005535A2 true WO2004005535A2 (en) | 2004-01-15 |
WO2004005535A3 WO2004005535A3 (en) | 2004-11-18 |
Family
ID=29763685
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2003/008209 WO2004005535A2 (en) | 2002-07-09 | 2003-07-09 | Pathogenicity proteins which can be used as targets for developing means for preventing and controlling bacterial infections |
Country Status (12)
Country | Link |
---|---|
US (3) | US20060003393A1 (en) |
EP (1) | EP1523572B1 (en) |
JP (1) | JP2006515155A (en) |
AT (1) | ATE394503T1 (en) |
AU (2) | AU2003266246A1 (en) |
CA (1) | CA2491847A1 (en) |
DE (1) | DE60320797D1 (en) |
DK (1) | DK1523572T3 (en) |
ES (1) | ES2306891T3 (en) |
FR (1) | FR2842210B1 (en) |
HK (1) | HK1078902A1 (en) |
WO (1) | WO2004005535A2 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006089264A3 (en) * | 2005-02-18 | 2007-04-05 | Novartis Vaccines & Diagnostic | Proteins and nucleic acids from meningitis/sepsis-associated escherichia coli |
EP2298795A1 (en) | 2005-02-18 | 2011-03-23 | Novartis Vaccines and Diagnostics, Inc. | Immunogens from uropathogenic escherichia coli |
EP2586790A2 (en) | 2006-08-16 | 2013-05-01 | Novartis AG | Immunogens from uropathogenic Escherichia coli |
US10058600B2 (en) | 2009-07-16 | 2018-08-28 | Glaxosmithkline Biologicals Sa | Detoxified Escherichia coli immunogens |
US11905286B2 (en) | 2018-08-09 | 2024-02-20 | Antabio Sas | Diazabicyclooctanones as inhibitors of serine beta-lactamases |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2842210B1 (en) * | 2002-07-09 | 2012-12-14 | Mutabilis | PATHOGENICITY DETERMINANTS FOR USE AS TARGETS FOR PREPARING AND CONTROLLING BACTERIAL INFECTIONS AND / OR SYSTEMIC DISSEMINATION |
WO2004050846A2 (en) * | 2002-12-02 | 2004-06-17 | Biosynexus Incorporated | Wall teichoic acid as a target for anti-staphylococcal therapies and vaccines |
EP1973467B1 (en) * | 2006-01-20 | 2013-10-16 | The General Hospital Corporation | Systems and process for providing speckle reduction using a wave front modulation for optical coherence tomography |
BR112018071867A2 (en) * | 2016-04-25 | 2019-02-19 | Aperture Bio Llc | flow cytometer method in an automatic fluid manipulation system, flow cytometer method for testing body fluid samples, inaccuracy compensation method of flow cytometer enumeration of particles of interest in fluid samples, and automatic body fluid sample testing system |
CN115725607B (en) * | 2022-07-14 | 2023-11-28 | 山东第一医科大学附属省立医院(山东省立医院) | Pathogenic target gene of nocardia melitensis and application thereof |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999066049A2 (en) * | 1998-06-12 | 1999-12-23 | University Of Guelph | A novel gene and method of treating a gram negative bacterial infection |
US6020121A (en) * | 1995-09-29 | 2000-02-01 | Microcide Pharmaceuticals, Inc. | Inhibitors of regulatory pathways |
WO2000028038A2 (en) * | 1998-11-09 | 2000-05-18 | Microscience Limited | Virulence genes and proteins, and their use |
WO2000044906A2 (en) * | 1999-01-27 | 2000-08-03 | Elitra Pharmaceuticals, Inc. | GENES IDENTIFIED AS REQUIRED FOR PROLIFERATION IN $i(ESCHERICHIA COLI) |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2842210B1 (en) * | 2002-07-09 | 2012-12-14 | Mutabilis | PATHOGENICITY DETERMINANTS FOR USE AS TARGETS FOR PREPARING AND CONTROLLING BACTERIAL INFECTIONS AND / OR SYSTEMIC DISSEMINATION |
-
2002
- 2002-07-09 FR FR0208636A patent/FR2842210B1/en not_active Expired - Lifetime
-
2003
- 2003-07-09 US US10/520,820 patent/US20060003393A1/en not_active Abandoned
- 2003-07-09 WO PCT/EP2003/008209 patent/WO2004005535A2/en active IP Right Grant
- 2003-07-09 CA CA002491847A patent/CA2491847A1/en not_active Abandoned
- 2003-07-09 DE DE60320797T patent/DE60320797D1/en not_active Expired - Lifetime
- 2003-07-09 DK DK03762684T patent/DK1523572T3/en active
- 2003-07-09 AU AU2003266246A patent/AU2003266246A1/en not_active Abandoned
- 2003-07-09 JP JP2004518773A patent/JP2006515155A/en active Pending
- 2003-07-09 EP EP03762684A patent/EP1523572B1/en not_active Expired - Lifetime
- 2003-07-09 ES ES03762684T patent/ES2306891T3/en not_active Expired - Lifetime
- 2003-07-09 AT AT03762684T patent/ATE394503T1/en active
-
2005
- 2005-10-20 HK HK05109367A patent/HK1078902A1/en not_active IP Right Cessation
-
2006
- 2006-08-21 US US11/506,821 patent/US20070111229A1/en not_active Abandoned
-
2009
- 2009-04-30 AU AU2009201727A patent/AU2009201727A1/en not_active Abandoned
-
2010
- 2010-03-23 US US12/729,630 patent/US20110236886A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6020121A (en) * | 1995-09-29 | 2000-02-01 | Microcide Pharmaceuticals, Inc. | Inhibitors of regulatory pathways |
WO1999066049A2 (en) * | 1998-06-12 | 1999-12-23 | University Of Guelph | A novel gene and method of treating a gram negative bacterial infection |
WO2000028038A2 (en) * | 1998-11-09 | 2000-05-18 | Microscience Limited | Virulence genes and proteins, and their use |
WO2000044906A2 (en) * | 1999-01-27 | 2000-08-03 | Elitra Pharmaceuticals, Inc. | GENES IDENTIFIED AS REQUIRED FOR PROLIFERATION IN $i(ESCHERICHIA COLI) |
Non-Patent Citations (46)
Title |
---|
ABACHIN ERIC ET AL: "Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes" MOLECULAR MICROBIOLOGY, vol. 43, no. 1, January 2002 (2002-01), pages 1-14, XP002284617 ISSN: 0950-382X * |
AUSTIN A E ET AL: "Genetic analysis of lipopolysaccharide core biosynthesis by Escherichia coli k12 insertion mutagenesis of the RFA locus" JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 172, no. 9, September 1990 (1990-09), pages 5312-5325, XP000926028 ISSN: 0021-9193 * |
BOYD E F & HARTL D L: "Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution" JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 180, no. 5, March 1998 (1998-03), pages 1159-1165, XP002133065 ISSN: 0021-9193 * |
BURNS STACY M ET AL: "Comparison of loss of serum resistance by defined lipopolysaccharide mutants and an acapsular mutant of uropathogenic Escherichia coli O75:K5." INFECTION AND IMMUNITY, vol. 66, no. 9, 1998, pages 4244-4253, XP002250632 ISSN: 0019-9567 cited in the application * |
CHANG HWAN-YOU ET AL: "Virulence and outer membrane properties of a galU mutant of Klebsiella pneumoniae CG43" MICROBIAL PATHOGENESIS, vol. 20, no. 5, 1996, pages 255-261, XP002284619 ISSN: 0882-4010 * |
CIESLEWICZ M ET AL: "THERMOREGULATION OF KPSF, THE FIRST REGION 1 GENE IN THE KPS LOCUS FOR POLYSIALIC ACID BIOSYNTHESIS IN ESCHERICHIA COLI K1" JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 178, no. 11, June 1996 (1996-06), pages 3212-3220, XP000877094 ISSN: 0021-9193 * |
DAINES DAYLE A ET AL: "NeuD plays a role in the synthesis of sialic acid in Escherichia coli K1" FEMS MICROBIOLOGY LETTERS, vol. 189, no. 2, 15 August 2000 (2000-08-15), pages 281-284, XP002284613 ISSN: 0378-1097 * |
DATABASE GENESEQ 2 July 2002 (2002-07-02), "DLTA" XP002284651 Database accession no. ABP30484 * |
DATABASE UNIPROT 1 February 1995 (1995-02-01), "KpsD" XP002284645 Database accession no. Q03961 * |
DATABASE UNIPROT 1 July 1993 (1993-07-01), "GmhB" XP002284647 Database accession no. P31546 * |
DATABASE UNIPROT 1 June 1994 (1994-06-01), "PGMU" XP002284644 Database accession no. P36938 * |
DATABASE UNIPROT 1 March 2002 (2002-03-01), "DltD" XP002284650 Database accession no. Q8VM64 * |
DATABASE UNIPROT 1 May 1992 (1992-05-01), "GalU" XP002284643 Database accession no. P25520 * |
DATABASE UNIPROT 1 May 1999 (1999-05-01), "WaaC" XP002284637 Database accession no. Q9ZITI1 * |
DATABASE UNIPROT 1 May 1999 (1999-05-01), "WaaO" XP002284642 Database accession no. Q9ZIS5 * |
DATABASE UNIPROT 1 May 2000 (2000-05-01), "WaaG" XP002284640 Database accession no. Q9R2L8 * |
DATABASE UNIPROT 1 May 2000 (2000-05-01), "WaaP" XP002284639 Database accession no. Q9R9D6 * |
DATABASE UNIPROT 1 May 2000 (2000-05-01), "WaaQ" XP002284638 Database accession no. Q9R9D5 * |
DATABASE UNIPROT 1 November 1990 (1990-11-01), "RFAD" XP002284648 Database accession no. P17963 * |
DATABASE UNIPROT 1 November 1996 (1996-11-01), "Neud" XP002284646 Database accession no. Q46674 * |
DATABASE UNIPROT 1 October 1994 (1994-10-01), "RFAF" XP002284641 Database accession no. P37692 * |
DATABASE UNIPROT 15 July 1999 (1999-07-15), "rfaE" XP002284649 Database accession no. P76658 * |
GENEVAUX PIERRE ET AL: "Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion" ARCHIVES OF MICROBIOLOGY, vol. 172, no. 1, July 1999 (1999-07), pages 1-8, XP002284584 ISSN: 0302-8933 * |
HEINRICHS DAVID E ET AL: "Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica." MOLECULAR MICROBIOLOGY, vol. 30, no. 2, October 1998 (1998-10), pages 221-232, XP002118873 ISSN: 0950-382X * |
HONG MEI ET AL: "Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance." MOLECULAR MICROBIOLOGY, vol. 24, no. 4, 1997, pages 779-791, XP008020612 ISSN: 0950-382X * |
KNEIDINGER BERND ET AL: "Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 24, 15 June 2001 (2001-06-15), pages 20935-20944, XP002284580 ISSN: 0021-9258 * |
KNEIDINGER BERND ET AL: "Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli" JOURNAL OF BACTERIOLOGY, vol. 184, no. 2, January 2002 (2002-01), pages 363-369, XP002284579 ISSN: 0021-9193 * |
LEE N G ET AL: "Molecular cloning and characterization of the nontypable Haemophilus influenzae-2019 rfaE gene required for lipopolysaccharide biosynthesis" INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY. WASHINGTON, US, vol. 63, no. 3, 1995, pages 818-824, XP000953326 ISSN: 0019-9567 * |
MARTINDALE J ET AL: "Genetic analysisof E. Coli K1 gastrointestinal colonization" MOLECULAR MICROBIOLOGY, vol. 37, no. 6, September 2000 (2000-09), pages 1293-1305, XP002284618 ISSN: 0950-382X * |
MERINO SUSANA ET AL: "Cloning and sequencing of the Klebsiella pneumoniae O5 wb gene cluster and its role in pathogenesis." INFECTION AND IMMUNITY, vol. 68, no. 5, May 2000 (2000-05), pages 2435-2440, XP002250634 ISSN: 0019-9567 * |
MOXON R ET AL: "CHALLENGE OF INVESTIGATING BILOGICALLY RELEVANT FUNCTIONS OF VIRULENCE FACTORS IN BACTERIAL PATHOGENS" PHILOSOPHICAL TRANSACTIONS. ROYAL SOCIETY OF LONDON. BIOLOGICAL SCIENCES, ROYAL SOCIETY, LONDON, GB, vol. 355, no. 1397, 25 May 2000 (2000-05-25), pages 643-656, XP001007306 ISSN: 0962-8436 * |
NESPER JUTTA ET AL: "Characterization of Vibrio cholerae O1 El Tor galU and galE mutants: Influence on lipopolysaccharide structure, colonization, and biofilm formation" INFECTION AND IMMUNITY, vol. 69, no. 1, January 2001 (2001-01), pages 435-445, XP002284616 ISSN: 0019-9567 * |
NGELEKA MUSANGU ET AL: "Characterization of a polysaccharide capsular antigen of septicemic Escherichia coli O115: K "V165": F165 and evaluation of its role in pathogenicity" INFECTION AND IMMUNITY, vol. 60, no. 12, 1992, pages 5048-5056, XP002284585 ISSN: 0019-9567 * |
RAUTEMAA RIINA ET AL: "Complement-resistance mechanisms of bacteria." MICROBES AND INFECTION, vol. 1, no. 10, August 1999 (1999-08), pages 785-794, XP002250635 ISSN: 1286-4579 cited in the application * |
REGNI CATHERINE ET AL: "Crystal structure of PMM/PGM: An enzyme in the biosynthetic pathway of P. aeruginosa virulence factors" STRUCTURE (CAMBRIDGE), vol. 10, no. 2, February 2002 (2002-02), pages 269-279, XP002284620 ISSN: 0969-2126 * |
RUSSO THOMAS A ET AL: "Loss of the O4 antigen moiety from the lipopolysaccharide of an extraintestinal isolate of Escherichia coli has only minor effects on serum sensitivity and virulence in vivo." INFECTION AND IMMUNITY, vol. 63, no. 4, 1995, pages 1263-1269, XP002250633 ISSN: 0019-9567 cited in the application * |
See also references of EP1523572A2 * |
SHEA JACQUELINE E ET AL: "Signature-tagged mutagenesis in the identification of virulence genes in pathogens." CURRENT OPINION IN MICROBIOLOGY, vol. 3, no. 5, October 2000 (2000-10), pages 451-458, XP002250636 ISSN: 1369-5274 * |
SISTI FEDERICO ET AL: "In vitro and in vivo characterization of a Bordetella bronchiseptica mutant strain with a deep rough lipopolysaccharide structure" INFECTION AND IMMUNITY, vol. 70, no. 4, April 2002 (2002-04), pages 1791-1798, XP002284581 ISSN: 0019-9567 * |
STOJILJKOVIC I ET AL: "CLONING AND CHARACTERIZATION OF THE NEISSERIA MENINGITIDIS RFAC GENE ENCODING ALPHA-1,5 HEPTOSYLTRANSFERASE I" FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, vol. 151, no. 1, 1997, pages 41-49, XP001007307 ISSN: 0378-1097 * |
TANG C ET AL: "PATHOGEN VIRULENCE GENES - IMPLICATIONS FOR VACCINES AND DRUG THERAPY" BRITISH MEDICAL BULLETIN, CHURCHILL LIVINGSTONE, LONDON, GB, vol. 55, no. 2, 1999, pages 387-400, XP008008098 ISSN: 0007-1420 * |
VALVANO M A ET AL: "The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose" JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 182, no. 2, January 2000 (2000-01), pages 488-497, XP000926030 ISSN: 0021-9193 * |
YETHON J A ET AL: "Involvement of waaY, waaQ and waaP in the modification of E. coli lipopolysaccharide and their role in the formation of a stable outer membrane" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 273, no. 41, 9 October 1998 (1998-10-09), pages 26310-26316, XP002118875 ISSN: 0021-9258 * |
YETHON JEREMY A ET AL: "Mutation of the lipopolysaccharide core glycosyltransferase encoded by waaG destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation" JOURNAL OF BACTERIOLOGY, vol. 182, no. 19, October 2000 (2000-10), pages 5620-5623, XP002284615 ISSN: 0021-9193 * |
YETHON JEREMY A ET AL: "Purification and characterization of Waap from Escherichia coli, a lipopolysaccharide kinase essential for outer membrane stability" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 8, 23 February 2001 (2001-02-23), pages 5498-5504, XP002284582 ISSN: 0021-9258 * |
YETHON JEREMY A ET AL: "Salmonella enterica serovar typhimurium waaP mutants show increased susceptibility to polymyxin and loss of virulence in vivo" INFECTION AND IMMUNITY, vol. 68, no. 8, August 2000 (2000-08), pages 4485-4491, XP002284583 ISSN: 0019-9567 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006089264A3 (en) * | 2005-02-18 | 2007-04-05 | Novartis Vaccines & Diagnostic | Proteins and nucleic acids from meningitis/sepsis-associated escherichia coli |
EP2298795A1 (en) | 2005-02-18 | 2011-03-23 | Novartis Vaccines and Diagnostics, Inc. | Immunogens from uropathogenic escherichia coli |
EP2351772A1 (en) | 2005-02-18 | 2011-08-03 | Novartis Vaccines and Diagnostics, Inc. | Proteins and nucleic acids from meningitis/sepsis-associated Escherichia coli |
US8758764B2 (en) | 2005-02-18 | 2014-06-24 | Novartis Vaccines And Diagnostics Srl | Proteins and nucleic acids from meningitis/sepsis-associated Escherichia coli |
US9334313B2 (en) | 2005-02-18 | 2016-05-10 | Glaxosmithkline Biologicals Sa | Proteins and nucleic acids from meningitis/sepsis-associated Escherichia coli |
US10035826B2 (en) | 2005-02-18 | 2018-07-31 | Glaxosmithkline Biologicals Sa | Proteins and nucleic acids from meningitis/sepsis-associated Escherichia coli |
EP2586790A2 (en) | 2006-08-16 | 2013-05-01 | Novartis AG | Immunogens from uropathogenic Escherichia coli |
US10058600B2 (en) | 2009-07-16 | 2018-08-28 | Glaxosmithkline Biologicals Sa | Detoxified Escherichia coli immunogens |
US11905286B2 (en) | 2018-08-09 | 2024-02-20 | Antabio Sas | Diazabicyclooctanones as inhibitors of serine beta-lactamases |
Also Published As
Publication number | Publication date |
---|---|
ES2306891T3 (en) | 2008-11-16 |
FR2842210A1 (en) | 2004-01-16 |
HK1078902A1 (en) | 2006-03-24 |
JP2006515155A (en) | 2006-05-25 |
AU2003266246A1 (en) | 2004-01-23 |
US20110236886A1 (en) | 2011-09-29 |
AU2009201727A1 (en) | 2009-05-21 |
FR2842210B1 (en) | 2012-12-14 |
WO2004005535A3 (en) | 2004-11-18 |
US20060003393A1 (en) | 2006-01-05 |
ATE394503T1 (en) | 2008-05-15 |
DE60320797D1 (en) | 2008-06-19 |
US20070111229A1 (en) | 2007-05-17 |
EP1523572A2 (en) | 2005-04-20 |
CA2491847A1 (en) | 2004-01-15 |
DK1523572T3 (en) | 2008-09-08 |
EP1523572B1 (en) | 2008-05-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20110236886A1 (en) | Pathogenicity determinants which can be used as targets for developing means for preventing and controlling bacterial infections and/or systemic dissemination | |
Timmis et al. | Surface components of Escherichia coli that mediate resistance to the bactericidal activities of serum and phagocytes | |
Skurnik et al. | A novel locus of Yersinia enterocolitica serotype O: 3 involved in lipopolysaccharide outer core biosynthesis | |
Keo et al. | Campylobacter capsule and lipooligosaccharide confer resistance to serum and cationic antimicrobials | |
Jones et al. | Identification of Streptococcus agalactiae virulence genes in the neonatal rat sepsis model using signature‐tagged mutagenesis | |
Chan et al. | Contributions of two-component regulatory systems, alternative σ factors, and negative regulators to Listeria monocytogenes cold adaptation and cold growth | |
JP2010207227A (en) | New antimicrobial polypeptide and method of use | |
Dentovskaya et al. | Functional characterization and biological significance of Yersinia pestis lipopolysaccharide biosynthesis genes | |
JP2010534063A (en) | Lantibiotics and their use | |
Zhang et al. | Novel Aeromonas hydrophila PPD134/91 genes involved in O-antigen and capsule biosynthesis | |
US8071356B2 (en) | Salmonella enterica strains of reduced pathogenicity, method for their preparation and uses thereof | |
CA2450658A1 (en) | Essential and important genes of pseudomonas aeruginosa and the use thereof to design or identify antibacterial agents | |
EP2253707A2 (en) | Expec-specific proteins, genes encoding them and uses thereof | |
Wooley et al. | Analysis of plasmids cloned from a virulent avian Escherichia coli and transformed into Escherichia coli DH5α | |
Lemos et al. | Stress responses of Streptococci | |
Saito et al. | Fate of Legionella pneumophila in macrophages of C57BL/6 chronic granulomatous disease mice | |
Hallenbeck et al. | The role of the universal sugar transport system components PtsI (EI) and PtsH (HPr) in Enterococcus faecium | |
Hoffman | The Role of the Transcriptional Antiterminator RfaH in Lipopolysaccharide Synthesis, Resistance to Antimicrobial Peptides, and Virulence of Yersinia Pseudotuberculosis and Yersinia Pestis | |
Chatzidaki | Relationship of the Vibrio vulnificus group 1-like capsular polysaccharide operon to phase variation | |
Vivo | efa1/Citrobacter rodentium lifA | |
Walker | Molecular characterization of IS1223 and localization of the insertion element to strategic positions in the genome of Lactobacillus johnsonii | |
Shippy et al. | Characterization of SEN3800-associated virulence of Salmonella enterica serovar Enteritidis phage type 8 | |
WO2003089572A2 (en) | Essential and important genes of pseudomonas aeroginosa and the use thereof to design or identify antibacterial agents | |
Hamilton et al. | In Vivo Gene Expression Analysis Identifies | |
by Macrophages et al. | Revised manuscript (IAI05693-11) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2003266246 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2491847 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2004518773 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2003762684 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2003762684 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2006003393 Country of ref document: US Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10520820 Country of ref document: US |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWP | Wipo information: published in national office |
Ref document number: 10520820 Country of ref document: US |
|
WWG | Wipo information: grant in national office |
Ref document number: 2003762684 Country of ref document: EP |