Moraxella catarrhalis protein, gene sequence and uses thereof

Details Moraxella catarrhalis protein, gene sequence and uses thereof Moraxella catarrhalis protein, gene sequence and uses thereof

1998-10-01

2003-04-01

Wilson, Michael C. (Department: 1632)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023740, C435S320100

Reexamination Certificate

active

06541616

ABSTRACT:

TABLE OF CONTENTS
1. FIELD OF THE INVENTION
2. BACKGROUND OF THE INVENTION
3. SUMMARY OF THE INVENTION
3.1. DEFINITIONS AND ABBREVIATIONS
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION OF THE INVENTION
5.1. OMP21 POLYPEPTIDE
5.2. OMP21-DERIVED POLYPEPTIDES
5.3. ISOLATION AND PURIFICATION OF OMP21
5.4. OMP21 IMMUNOGENS AND ANTI-OMP21 ANTIBODIES
5.5. PHARMACEUTICAL COMPOSITIONS
5.6. METHODS OF DETECTING
5.7. NUCLEIC ACIDS ENCODING OMP21
5.8. RECOMBINANT PRODUCTION OF OMP21
5.9. APPLICATIONS
6. EXAMPLE: ISOLATION AND CHARACTERIZATION OF THE OMP21 POLYPEPTIDE AND GENE ENCODING SAME
6.1. MATERIALS AND METHODS
6.1.1. DETERGENT EXTRACTION OF OMP21
6.1.2. AMINO TERMINAL SEQUENCING OF OMP21
6.1.3. ANTI-OMP21 ANTISERUM
6.1.4. WESTERN BLOTS
6.1.5. OUTER SURFACE LOCALIZATION OF OMP21
6.2. RESULTS
6.2.1. OUTER SURFACE LOCATION OF OMP21
6.2.2. PROPERTIES OF OMP21 POLYPEPTIDE
6.2.3. CONSERVATION OF OMP21 POLYPEPTIDE
7. EXAMPLE: EFFICACY OF OMP21 VACCINE: CYTOTOXIC ACTIVITY OF ANTI-OMP21 ANTISERUM
8. EXAMPLE: ISOLATION OF THE omp21 GENE
8.1. PREPARATION OF PRIMERS
8.2. SUPPRESSION PCR
8.3. ISOLATION AND SUBCLONING OF THE PCR PRODUCT
8.4. IDENTIFICATION OF RECOMBINANT PLASMIDS
8.5. SEQUENCE ANALYSIS
9. EXAMPLE: PREPARATION OF RECOMBINANT OMP21
9.1. CONSTRUCTION OF AN EXPRESSION VECTOR
9.2. EXPRESSION OF OMP21
10. EXAMPLE: VERIFICATION OF THE omp21 GENE
10.1. CONSTRUCTION OF AN omp21 GENE-TARGETING CASSETTE
10.2 PREPARATION OF COMPETENT MORAXELLA CATARRHALIS CELLS
10.3. ELECTROPORATION OF COMPETENT CELLS
10.4. PCR ANALYSIS OF PUTATIVE OMP21 DELETION MUTANTS
10.5. SOUTHERN ANALYSIS OF OMP 21 DELETION MUTANTS
11. EXAMPLE: RFLP ANALYSIS OF omp 21
12. EXAMPLE: GENERATION AND REACTIVITY OF MONOCLONAL ANTI-OMP21 ANTIBODIES
13. EXAMPLE: NASOPHARYNGEAL CELL BINDING
14. DEPOSIT OF MICROORGANISM
1. FIELD OF THE INVENTION
The present invention generally relates to an isolated or substantially purified protein obtainable from the outer membranes of
M. catarrhalis,
called “OMP21” (defined below in Section 3). The invention also encompasses the amino acid sequence thereof, and antibodies, including cytotoxic antibodies, that specifically bind OMP21. The invention further encompasses pharmaceutical compositions, including prophylactic or therapeutic compositions, and which may be immunogenic compositions, including vaccines. The invention additionally provides methods of preventing, treating or ameliorating disorders in mammals related to
M. catarrhalis
infections and for inducing immune responses to
M. catarrhalis.
The invention further provides isolated nucleotide sequences encoding the OMP21, homologous and complementary sequences thereto, vectors having said sequences, host cells containing said vectors, and prophylactic or therapeutic compositions, which may be immunogenic compositions, including vaccines comprising same. Diagnostic methods and kits are also included.
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 humans. 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 (Helminen et al., 1993, Infect. Immun. 61:2003-2010; Catlin; B. W., 1990, Clin. Microbiol. Rev. 3:293-320; and references cited therein). 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 over 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 98 to 21 kD, respectively (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 polyarylamide 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 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 surface-exposed epitopes of outer-membrane proteins of
M. catarrhalis
is generally examined by the cytotoxic (bactericidal) activity, because there is no animal model of disease. The only natural host for disease caused by Moraxella is human. However, others have studied the role of antibodies in an animal model of Moraxella lung clearance. 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, all of which are relatively rapid, 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 CopB and 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). There remains a need for compositions and methods for diagnosis of, as well as, prophylactic and therapeutic treatments for infections caused by
M. catarrhalis.
The adherence of bacterial pathogens to a host cell surface promotes colonization and initiates pathogenesis. See, E. H. Beachey, 1981, J. Infect. Dis. 143:325-345. Gram-negative bacteria typically express surface lectins that bind to specific oligosaccharides of glycoproteins and/or glycolipids on the host cell surface. Such lectins are often associated with pili or fimbriae. Bacterial adherence can also occur by non-specific binding resulting from hydrophobic and/or charge interaction with the host cell surface.
The mechanism of
M. catarrhalis
adherence to cells of the respiratory tract remains poorly understood. The organism adheres to cultured human nasopharyngeal epithelial cells. Another study suggests that fimbriae may have a role in the adherence to such cells as fimbriae denaturation or treatment with anti-fimbriae antibodies reduced adherence by fimbriated strains. Fimbriae mediated binding, however, cannot be the sole basis of this adherence as the most highly adhering strain, among the several examined, was a non-fimbriated strain. Thus, other unidentified components are involved in the bacteria's adherence.
3. SUMMARY OF THE INVENTION
An object of the present invention is to provide an isolated or substantially purified OMP21 protein of a
M. catarrhalis
strain, wherein the apparent molecular weight is about 16 kD to about 20 kD, as predicted from the deduced amino acid sequence or determined by sodium dodecylsulfate polyacrylamide gel e

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