Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-11-12
2001-04-10
Smith, Lynette R. F. (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023700, C424S184100, C424S190100, C424S234100
Reexamination Certificate
active
06214981
ABSTRACT:
TABLE OF CONTENTS
1. INTRODUCTION
2. BACKGROUND OF THE INVENTION
2.1. OUTER MEMBRANE PROTEINS AND PROTECTIVE ANTIBODIES
2.2. BACTERIAL/HOST CELL ADHERENCE AND HEMAGGLUTINATION
3. SUMMARY OF THE INVENTION
3.1. DEFINITIONS AND ABBREVIATIONS
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. HEMAGGLUTINATING AND NON-HEMAGGLUTINATING CULTIVARS
5.2. OMP106 POLYPEPTIDE
5.3. OMP106-DERIVED POLYPEPTIDES
5.4. ISOLATION AND PURIFICATION OF OMP106 POLYPEPTIDE AND OMP106-DERIVED POLYPEPTIDES
5.5. OMP106 IMMUNOGENS AND ANTI-OMP106 ANTIBODIES
5.6. VACCINES
5.7. NUCLEIC ACIDS ENCODING OMP106 POLYPEPTIDE AND OMP106-DERIVED POLYPEPTIDES
5.8. RECOMBINANT PRODUCTION OF OMP106 POLYPEPTIDE AND OMP106-DERIVED POLYPEPTIDES
5.9. REAGENTS
6. EXAMPLE: ISOLATION AND CHARACTERIZATION OF THE OMP106 POLYPEPTIDE AND GENE ENCODING SAME FROM STRAIN ATCC 49143 OR OTHER STRAINS
6.1. MATERIAL AND METHODS
6.1.1. HEMAGGLUTINATION ASSAY
6.1.2. INHIBITION OF HEMAGGLUTINATION
6.1.3. LIGAND AND RECEPTOR BINDING
6.1.4. OG EXTRACTION OF OMPS
6.1.5. PROTEOLYTIC DIGESTION OF OMP106
6.1.6. NON-HEMAGGLUTINATING CULTIVARS
6.1.7. ISOLATION OF OMP106 POLYPEPTIDE
6.1.8. ANTI-OMP106 ANTISERUM
6.1.9. WESTERN BLOTS WITH ANTI-OMP106 ANTISERUM
6.1.10. ANTI-OMP106 IMMUNOFLUORESCENCE STAINING OF CELL SURFACE
6.2. RESULTS
6.2.1. HEMAGGLUTINATION ACTIVITY
6.2.2. OMP106 RECEPTORS AND LIGANDS
6.2.3. IDENTIFICATION OF OMP106 POLYPEPTIDE
6.2.4. OMP PROFILE OF NHA CULTIVARS
6.2.5. OMP106 AND HEMAGGLUTINATION
6.2.6. OUTER SURFACE LOCATION OF OMP106
6.2.7. PROPERTIES OF OMP106 POLYPEPTIDE
6.2.8. CONSERVATION OF OMP106 POLYPEPTIDE
7. EXAMPLE: EFFICACY OF OMP106 VACCINE: CYTOTOXIC ACTIVITY OF ANTI-OMP106 ANTISERUM
8. EXAMPLE: ISOLATION OF THE omp 106 GENE
8.1. PREPARATION OF 72 BP PRIMER
8.2. PCR AMPLIFICATION OF A 72 BP DNA FRAGMENT FROM
MORAXELLA CATARRHALIS
GENOMIC DNA
8.3. ISOLATION AND SUBCLONING OF THE 72 BP PCR AMPLIFICATION PRODUCT
8.4. SEQUENCING OF THE 72 BP INSERT IN PCR SCRIPT AMP SK(+)
8.5. GENERATION OF A RADIOLABELED 72 BP DNA SCREENING PROBE
8.6. HYBRIDIZATION OF PLAQUE-LIFT FILTERS AND SOUTHERN BLOTS WITH RADIOLABELED PROBE
8.7. CONSTRUCTION OF A
MORAXELLA CATARRHALIS
GENOMIC DNA LIBRARY
8.8. LARGE SCALE PHAGE DNA ISOLATION
8.9. DETERMINATION OF INSERT SIZE AND MAPPING OF DNA FRAGMENTS HYBRIDIZING WITH THE 72 BP PROBE
8.10. MAPPING OF THE 72 BP DNA REGION IN THE SUBCLONED NOTI/PSTI FRAGMENT OF omp106.6
8.11. MAPPING OF THE 12 KB HINDIII INSERT FROM omp106.6
8.12. SUBCLONING OF RESTRICTION FRAGMENTS OF omp106.6 AND ANALYSIS OF RECOMBINANT PLASMIDS
9. EXAMPLE: SEQUENCING OF THE omp 106 GENE
10. EXAMPLE: VERIFICATION OF THE omp 106 GENE
10.1. CONSTRUCTION OF AN omp 106 GENE-TARGETING CASSETTE
10.2. PREPARATION OF ELECTROCOMPETENT
MORAXELLA CATARRHALIS
CELLS
10.3. ELECTROPORATION OF COMPETENT CELLS
11. EXAMPLE: GENETIC ANALYSIS
11.1. PCR ANALYSIS
11.2. SOUTHERN ANALYSIS OF omp 106
12. EXAMPLE: GENERATION AND REACTIVITY OF MONOCLONAL ANTI-OMP106 ANTIBODIES
13. EXAMPLE: BINDING AND INHIBITION OF BINDING TO NASOPHARYNGEAL CELLS
13.1. NASOPHARYNGEAL CELL BINDING
13.2. INHIBITION OF NASOPHARYNGEAL BINDING
14. EXAMPLE: HEMAGGLUTINATION ASSAY
15. DEPOSIT OF MICROORGANISM
1. INTRODUCTION
The present invention generally relates to the outer membrane protein-106 (OMP106) polypeptide of
Moraxella catarrhalis
. The invention encompasses a purified OMP106 polypeptide and polypeptides derived therefrom (OMP106-derived polypeptides). The invention also encompasses antibodies, including cytotoxic antibodies, that specifically bind the OMP106 polypeptide and/or OMP106-derived polypeptides. The invention further encompasses prophylactic or therapeutic compositions, including vaccines, that comprise OMP106 polypeptide and/or OMP106-derived polypeptides. The invention additionally provides methods of inducing immune responses to
M. catarrhalis
in mammals. The invention further provides isolated nucleotide sequences encoding the OMP106 polypeptide and OMP106-derived polypeptides, vectors having said sequences, and host cells containing said vectors.
2. BACKGROUND OF THE INVENTION
Moraxella catarrhalis
, also known as
Moraxella
(
Branhamella
)
catarrhalis
or
Branhamella catarrhalis
and formerly known as
Neisseria catarrhalis
or
Micrococcus catarrhalis
, is a gram-negative bacterium frequently found in the respiratory tract of humans.
M. catarrhalis
, originally thought to be a harmless commensal organism, is now recognized as an important pathogen in upper and lower respiratory tract infections in animals. In humans,
M. catarrhalis
causes serious lower respiratory tract infections in adults with chronic lung disease, systemic infections in immunocompromised patients, and otitis media and sinusitis in infants and children. See Helminen et al., 1993, Infect. Immun. 61:2003-2010; Catlin, B. W., 1990, Clin. Microbiol. Rev. 3:293-320; and references cited therein.
2.1. OUTER MEMBRANE PROTEINS AND PROTECTIVE ANTIBODIES
The outer surface components of
Moraxella catarrhalis
have been studied in attempts to understand the pathogenic process of
M. catarrhalis
infections and to develop useful therapeutic treatments and prophylactic measures against such infections. The outer membrane proteins (OMPs) in particular have received considerable attention as possible virulence factors and as potential vaccine antigens.
M. catarrhalis
has about 10 to 20 different OMPs with 6 to 8 of these, OMPs A to H, as the predominate species (Murphy and Loeb, 1989, Microbial Pathogen. 6:159-174). The molecular weights of OMPs A to H range from 97 to 20 kD, respectively. See Bartos and Murphy, 1988, J. Infect. Dis. 158:761-765; Helminen et al., 1993, Infect. Immun. 61:2003-2010; Murphy et al, 1993, Molecul. Microbiol. 10: 87-97; and Sarwar et al, 1992, Infect. Immun. 60:804-809. Comparisons of protein profiles by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane preparations from 50
M. catarrhalis
strains show nearly homogeneous patterns of OMPs A to H (Bartos and Murphy, 1988, J. Infect. Dis. 158:761-765).
In addition to OMPs A to H, a high molecular weight OMP, designated HMW-OMP, having an apparent mass of 350 to 720 kD by SDS-PAGE has also been identified as another prominent surface component present in many strains of
M. catarrhalis
. HMW-OMP upon formic acid denaturation produces a single band of 120 to 140 kD and, thus, appears to be an oligomeric protein (Klingman and Murphy, 1994, Infect. Immun. 62:1150-1155). HMW-OMP appears to be the same protein as that designated UspA by Helminen et al., (1994, J. Infect. Dis. 170:867-872) and shown to be present in a number of
M. catarrhalis
strains.
In intact bacterium or bacterially-derived outer membrane vesicles, several of the above-identified OMPs present surface-exposed epitopes that elicit the production of antibodies that bind the OMPs. These antigenic OMPs include OMP E and OMP G (Murphy and Bartos, 1989, Infect. Immun. 57:2938-2941); OMP C/D (Sarwar et al., 1992, Infect. Immun. 60:804-809); CopB, an 80 kD OMP, (Helminen et al., 1993, Infect. Immun. 61:2003-2010); and UspA (Helminen et al., 1994, J. Infect. Dis. 170:867-872).
The therapeutic potential of antibodies to surfaced-exposed epitopes of CopB and UspA has been evaluated in an animal model. The model involved direct bolus inoculation of lungs of BALB/c VAF/Plus mice with a controlled number of
M. catarrhalis
cells and subsequent examination of the rate of pulmonary clearance of the bacteria (Unhanand et al., 1992, J. Infect. Dis. 165:644-650). Different clinical isolates of the
M. catarrhalis
exhibited different rates of clearance that correlated with the level of granulocyte recruitment into the infection site. Passive immunization with a monoclonal antibody directed to a surface-exposed epitope of either CopB or UspA increased the rate of pulmonary clearance of
M. catarrhalis
(Helminen et al., 1993, Infect. Immun. 61:2003-2010; Helminen et al., 1994, J. Infect. Dis. 170:867-872).
2.2. BACTERIAL/HOST CELL ADHERENCE AND HEMAGGLUTINATION
The adherence of bacteri
Plosila Laura
Tillman Ulrich F.
Tucker Kenneth
Antex Biologics Inc.
Pennie & Edmonds LLP
Portner Ginny Allen
Smith Lynette R. F.
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