High molecular weight major outer membrane protein of moraxella

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...

Reexamination Certificate

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C424S184100, C424S185100, C424S190100, C424S234100, C424S803000, C530S300000, C530S324000, C530S325000, C530S326000

Reexamination Certificate

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06440425

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of immunology and is particularly concerned with outer membrane proteins from Moraxella, methods of production thereof, genes encoding such proteins and uses thereof.
BACKGROUND OF THE INVENTION
Otitis media is the most common illness of early childhood with approximately 70% of all children suffering at least one bout of otitis media before the age of seven. Chronic otitis media can lead to hearing, speech and cognitive impairment in children. It is caused by bacterial infection with
Streptococcus pneumoniae
(approximately 50%), non-typable
Haemophilus influenzae
(approximately 30%) and
Moraxella
(Branhamella)
catarrhalis
(approximately 20%). In the United States alone, treatment of otitis media costs between one and two billion dollars per year for antibiotics and surgical procedures, such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. Because otitis media occurs at a time in life when language skills are developing at a rapid pace, developmental disabilities specifically related to learning and auditory perception have been documented in youngsters with frequent otitis media.
M. catarrhalis
mainly colonizes the respiratory tract and is predominantly a mucosal pathogen. Studies using cultures of middle ear fluid obtained by tympanocentesis have shown that
M. catarrhalis
causes approximately 20% of cases of otitis media (ref. 1—Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure).
The incidence of otitis media caused by
M. catarrhalis
is increasing. As ways of preventing otitis media caused by pneumococcus and non-typable
H. influenzae
are developed, the relative importance of
M. catarrhalis
as a cause of otitis media can be expected to further increase.
M. cattarrhalis
is also an important cause of lower respiratory tract infections in adults, particularly in the setting of chronic bronchitis and emphysema (refs. 2, 3, 4, 5, 6, 7, and 8).
M. cattarrhalis
also causes sinusitis in children and adults (refs. 9, 10. 11, 12, and 13) and occasionally causes invasive disease (refs. 14, 15, 16, 17, 18, and 19).
Like other Gram-negative bacteria, the outer membrane of
M. cattarrhalis
consists of phospholipids, lipopolysaccharide (LPS), and outer membrane proteins (OMPs). Eight of the
M. cattarrhalis
OMPs have been identified as major components. These are designated by letters A to H, beginning with OMP A which has a molecular mass of 98 kDa to OMP H which has a molecular mass of 21 kDa (ref. 20).
Recently, a high-molecular-weight outer membrane protein of
M. cattarrhalis
was purified and characterized (ref. 21). The apparent molecular mass of this protein varies from 350 kDa to 720 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein appears to be an oligomer of much smaller proteins or subunits thereof of molecular mass 120 to 140 kDa and is antigenically conserved among strains of Moraxella.
A protein molecular mass of about 300 to 400 kDa named UspA was also reported to be present on the surface of Moraxella (ref. 22).
M. cattarrhalis
infection may lead to serious disease. It would be advantageous to provide other outer membrane proteins for
M. cattarrhalis
and genes encoding such proteins for use as antigens in immunogenic preparations including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents.
SUMMARY OF THE INVENTION
The present invention is directed towards the provision of a purified and isolated major outer membrane protein of
Moraxella catarrhalis
and other Moraxella strains, having an apparent molecular mass of about 200 kDa, as well as genes encoding the same.
In accordance with one aspect of the invention, there is provided an isolated and purified, outer membrane protein of a Moraxella strain having a molecular weight of about 200 kDa, as determined by SDS-PAGE, or a fragment or an analog thereof. The outer membrane protein may be substantially in its native conformation (so as to have substantially retained the characteristic immunogenicity of the outer membrane protein in the Moraxella strain) and may be isolated from a
M. catarrhalis
strain, such as from
M. cattarrhalis
4223. Such isolated and purified about 200 kDa outer membrane protein is substantially free from non-200 kDa outer membrane proteins, phospholipids and lipopolysaccharide of Moraxella. The about 200 kDa outer membrane protein is at least about 70 wt % pure, preferably at least about 90 wt % pure, and may be in the form of an aqueous solution thereof. Such about 200 kDa outer membrane protein may have substantially the amino acid composition shown in Table III and a deduced amino acid sequence as shown in
FIG. 6
(SEQ ID No: 3).
The present invention also provides a purified and isolated nucleic acid molecule encoding an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or a fragment or an analog of the outer membrane protein. The protein encoded by the nucleic acid molecule may comprise a protein containing the amino acid sequence NH
2
-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile (SEQ ID No: 5) particularly where X is Lys (SEQ ID No: 8), for
Moraxella catarrhalis
strain 4223 or containing the corresponding amino acid sequence from other Moraxella strains.
In a further aspect of the present invention, there is provided a purified and isolated nucleic acid molecule having a sequence selected from the group consisting of (a) a DNA sequence as set out in
FIG. 6
(SEQ ID Nos: 1 or 2), or the complementary sequence thereto; (b) a DNA sequence encoding an about 200 kDa protein of a strain of Moraxella and containing the amino acid sequence NH
2
-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile (SEQ ID No: 5), particularly where x is Lys (SEQ ID No: 8) or the complementary sequence thereto; (c) a DNA sequence encoding the deduced amino acid sequence as set out in
FIG. 6
(SEQ ID No: 3) or the complementary sequence thereto; and (d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c). The nucleic acid preferably defined in (d) has at least about 90% sequence identity with any one of the sequences defined in (a), (b) or (c).
The nucleic acid molecules provided herein may be included in a vector adapted for transformation of a host. The nucleic acid molecules provided herein also may be included in an expression vector adapted for transformation of a host along with expression means operatively coupled to the nucleic acid molecule for expression by the host of the about 200 kDa outer membrane protein of a strain of Noraxella or the fragment or the analog of the outer membrane protein. A transformed host containing the expression vector is included within the invention, along with a recombinant outer membrane protein or fragment or analog thereof producible by the transformed host.
The expression means may include a nucleic acid portion encoding a leader sequence for secretion from the host of the outer membrane protein or the fragment or the analog of the outer membrane protein. The expression means may include a nucleic acid portion encoding a lipidation signal for expression from the host of a lipidated form of the outer membrane protein or the fragment or analog thereof.
The present invention further includes a live vector for delivery of the outer membrane protein of the invention or a fragment or analog thereof, comprising a vector containing the nucleic acid molecule provided herein. The live vector may be selected from the group consisting of
E. coli,
Salmonell

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