Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-04-14
2002-08-27
Pak, Michael (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S069300, C435S069700, C435S071100, C435S071200, C435S252100, C435S252300, C435S325000, C536S023100, C536S023400, C536S023700
Reexamination Certificate
active
06440701
ABSTRACT:
FIELD OF INVENTION
The present invention relates to the molecular cloning of genes encoding transferrin receptor (TfR) proteins and, in particular, to the cloning of transferrin receptor 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. In recent years,
M. catarrhalis
has been recognized as an important causative agent of otitis 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).
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
commonly is 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 75% of clinical isolates produce &bgr;-lactamase (ref. 16, 26).
Iron is an essential nutrient for the growth of many bacteria. Several bacterial species, including
M. catarrhalis
, obtain iron from the host by using transferrin receptor proteins to capture transferrin. A number of bacteria including
Neisseria meningitidis
(ref. 17),
N. gonorrhoeae
(ref. 18),
Haemophilus influenzae
(ref. 19), as well as
M. catarrhalis
(ref. 20), produce outer membrane proteins which specifically bind human transferrin. The expression of these proteins is regulated by the amount of iron in the environment.
The two transferrin receptor proteins of
M. catarrhalis
, designated transferrin binding protein 1 (Tbp1) and transferrin binding protein 2 (Tbp2), have molecular weights of 115 kDa (Tbp1) and approximately 80 to 90 kDa (Tbp2). Unlike the transferrin receptor proteins of other bacteria which have an affinity for apotransferrin, the
M. catarrhalis
Tbp2 receptors have a preferred affinity for iron-saturated (i.e., ferri-) transferrin (ref. 21).
M. catarrhalis
infection may lead to serious disease. It would be advantageous to provide a recombinant source of transferrin binding proteins as antigens in immunogenic preparations including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents. The genes encoding transferrin 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 transferrin receptor of a strain of Moraxella or a fragment or an analog of the transferrin receptor protein. The nucleic acid molecules 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 tbp genes by recombinant DNA means for providing, in an economical manner, purified and isolated transferrin receptor proteins as well as subunits, fragments or analogs thereof. The transferrin 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 transferrin 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 transferrin receptor protein of a strain of Moraxella, more particularly, a strain of
M. catarrhalis
, specifically
M. catarrhalis
strain 4223, Q8, R1, M35, 3 or LES1, or a fragment or an analog of the transferrin receptor protein.
In one preferred embodiment of the invention, the nucleic acid molecule may encode only the Tbp1 protein of the Moraxella strain or only the Tbp2 protein of the Moraxella strain. In another preferred embodiment of the invention, the nucleic acid may encode a fragment of the transferrin receptor protein of a strain of Moraxella having an amino acid sequence which is conserved.
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. 5
,
6
,
10
,
11
,
27
,
31
,
32
or
33
(SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 45, 47, 48, 50 or 52 or the complementary DNA sequence thereto; (b) a DNA sequence encoding an amino acid sequence as set out in
FIG. 5
,
6
,
10
,
11
,
27
,
31
,
32
or
33
(SEQ ID NOS: 9, 10, 11, 12, 13, 14, 15, 16, 46, 49, 51 or 53 or the complementary DNA sequence thereto; and (c) a DNA sequence encoding a functional transferrin receptor protein of a strain 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) and (b). The functional transferrin receptor protein of a strain of Moraxella encoded by the DNA sequence defined in (c) is the equivalent transferrin receptor protein from another strain of Moraxella.
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 LEM3-24, pLEM3, pLEM25, pLEM23, SLRD-A, DS-1698-1-1, DS-1754-1, pSLRD2, pSLRD3, pSLRD4 and pSLRD5.
The vector may be adapted for expression of the encoded transferrin receptor, fragments or analogs thereof, in a
Du Run-Pan
Harkness Robin E.
Klein Michel H.
Loosmore Sheena M.
Myers Lisa E.
Aventis Pasteur Limited
Pak Michael
Sim & McBurney
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