Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-05-08
2001-02-06
Graser, Jennifer (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024300, C536S024320, C435S320100, C435S069100, C435S069300, C435S069700, C435S252300, C424S200100, C424S251100
Reexamination Certificate
active
06184371
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the molecular cloning of genes encoding lactoferrin receptor (LfR) proteins and, in particular, to the cloning of lactoferrin binding protein genes (lbp genes) from
Moraxella
(
Branhamella
)
catarrhalis.
BACKGROUND OF THE INVENTION
Moraxella
(
Branhamella
)
catarrhalis
bacteria are Gram-negative diplococcal pathogens which are carried asymptomatically in the healthy human respiratory tract. However, in recent years,
M. catarrhalis
has been recognized as an important causative agent of otilis media. In addition,
M. catarrhalis
has been associated with sinusitis, conjunctivitis, and urogenital infections, as well as with a number of inflammatory diseases of the lower respiratory tract in children and adults, including pneumonia, chronic bronchitis, tracheitis, and emphysema (refs. 1 to 8). (Throughout this application, various references are cited in parentheses to describe more fully 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). Occasionally,
M. catarrhalis
invades to cause septicaemia, arthritis, endocarditis, and meningitis (refs. 9 to 13).
M. catarrhalis
colonizes the human upper respiratory tract and is an important cause of otitis media in infants and children as well as lower respiratory tract infections in adults with chronic obstructive pulmonary disease.
Otitis media is one of the most common illnesses of early childhood and approximately 80% of all children suffer at least one middle ear infection before the age of three (ref. 14). Chronic otitis media has been associated with auditory and speech impairment in children, and in some cases, has been associated with learning disabilities. Conventional treatments for otitis media include antibiotic administration and surgical procedures, including tonsillectomies, adenoidectomies, and tympanocentesis. In the United States, treatment costs for otitis media are estimated to be between one and two billion dollars per year.
In otitis media cases,
M. catarrhalis
is commonly co-isolated from middle ear fluid along with
Streptococcus pneumoniae
and non-typable
Haemophilus influenzae
, which are believed to be responsible for 50% and 30% of otitis media infections, respectively.
M. catarrhalis
is believed to be responsible for approximately 20% of otitis media infections (ref. 15). Epidemiological reports indicate that the number of cases of otitis media attributable to
M. catarrhalis
is increasing, along with the number of antibiotic-resistant isolates of
M. catarrhalis
. Thus, prior to 1970, no &bgr;-lactamase-producing
M. catarrhalis
isolates had been reported, but since the mid-seventies, an increasing number of &bgr;-lactamase-expressing isolates have been detected. Recent surveys suggest that up to 80 to 85% of clinical isolates produce &bgr;-lactamase (ref. 16, 22, 23).
Iron-restriction is a general host defence mechanism against microbial pathogens. A number of bacterial species including
Neisseria meningitidis
(ref. 17, 24),
N. gonorrhoeae
(ref. 25) and
M. catarrhalis
(ref. 17), express outer membrane proteins which specifically bind human lactoferrin.
M. catarrhalis
infection may lead to serious disease. It would be advantageous to provide a recombinant source of lactoferrin binding proteins as antigens in immunogenic preparations including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents. The genes encoding lactoferrin binding proteins and fragments thereof are particularly desirable and useful in the specific identification and diagnosis of Moraxella and for immunization against disease caused by
M. catarrhalis
and for the generation of diagnostic reagents.
SUMMARY OF THE INVENTION
The present invention is directed towards the provision of purified and isolated nucleic acid molecules encoding a lactoferrin receptor protein of a strain of Moraxella or a fragment or an analog of the lactoferrin receptor protein. The nucleic acid molecules and isolated and purified lactoferrin binding proteins provided herein are useful for the specific detection of strains of Moraxella and for diagnosis of infection by Moraxella. The purified and isolated nucleic acid molecules provided herein, such as DNA, are also useful for expressing the lbp genes by recombinant DNA means for providing, in an economical manner, purified and isolated lactoferrin receptor proteins free of other Moraxella proteins, as well as subunits, fragments or analogs thereof.
The lactoferrin receptor, subunits or fragments thereof or analogs thereof, as well as nucleic acid molecules encoding the same and vectors containing such nucleic acid molecules, are useful in immunogenic compositions for vaccinating against diseases caused by Moraxella, the diagnosis of infection by Moraxella, and as tools for the generation of immunological reagents.
Monoclonal antibodies or mono-specific antisera (antibodies) raised against the lactoferrin receptor protein produced in accordance with aspects of the present invention are useful for the diagnosis of infection by Moraxella, the specific detection of Moraxella (in, for example, in vitro and in vivo assays) and for the treatment of diseases caused by Moraxella.
In accordance with one aspect of the present invention, there is provided a purified and isolated nucleic acid molecule encoding a lactoferrin receptor protein of a strain of Moraxella, more particularly a strain of
M. catarrhalis
, specifically
M. catarrhalis
strain 4223, Q8 or VH19 or a fragment or an analog of the lactoferrin receptor protein. A fragment of the lactoferrin receptor protein is a portion of the protein which retains the immunological properties of the protein.
In one preferred embodiment of the invention, the nucleic acid molecule may encode only the Lbp1 protein of the Moraxella strain or only the Lbp2 protein of the Moraxella strain or only the ORF3 protein of the Moraxella strain. In another preferred embodiment of the invention, the nucleic acid may encode a fragment of the lactoferrin receptor protein of a strain of Moraxella having a conserved amino acid sequence.
In a further aspect of the present invention, there is provided an isolated and purified nucleic acid molecule encoding at least one lactoferrin binding protein of Moraxella having a restriction map as shown in
FIG. 3
for
M. catarrhalis
4223,
FIG. 5
for
M. catarrhalis
Q8 or
FIG. 17
for
M. catarrhalis
VH19 or the equivalent map from other strains of Moraxella.
In another aspect of the present invention, there is provided a purified and isolated nucleic acid molecule having a DNA sequence selected from the group consisting of (a) a DNA sequence as set out in
FIG. 2
or
4
(SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 69) or the complementary DNA sequence thereto; (b) a DNA sequence encoding an amino acid sequence as set out in
FIG. 2
or
4
(SEQ ID Nos. 11, 12, 13, 14, 15, 16,17, 18, 70) or the complementary DNA sequence thereto; and (c) a DNA sequence encoding a functional lactoferrin receptor protein of Moraxella, which may be a. DNA sequence which hybridizes under stringent conditions to any one of the DNA sequences defined in (a) or (b). The DNA sequence defined in (c) may have at least about 90% sequence identity with any one of the DNA sequences defined in (a) or (b). Stringent conditions of hybridization are described below. Sequence identity is determined in the manner described below.
In an additional aspect, the present invention includes a vector adapted for transformation of a host, comprising a nucleic acid molecule as provided herein and may have the characteristics of a nucleotide sequence contained within vectors pLD3, pLDW3, PLD1-8 and pLDW1.
The vector may be adapted for expression of the encoded lactoferrin receptor protein, fragments or analogs thereof, in a heterologous or homologous ho
Du Run-Pan
Klein Michel H.
Loosmore Sheena M.
Wang Quijun
Yang Yan-Ping
Connaught Laboratories Limited
Graser Jennifer
Sim & McBurney
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