Transferrin receptor 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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C530S387100, C530S412000, C530S417000, C435S007100, C435S007800, C435S070200

Reexamination Certificate

active

06190668

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of immunology and is particularly concerned with transferrin receptor protein from Moraxella, methods of production, and uses thereof.
BACKGROUND OF THE INVENTION
Otitis media is the most common illness of early childhood with approximately 80% of all children suffering at least one bout of otitis media before the age of three (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). 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. 2).
Long regarded as an opportunistic pathogen,
B. catarrhalis
is now recognized to cause a variety of potentially debilitating human diseases as a result of localized or, more rarely, systemic infection.
M. catarrhalis
is an important cause of lower respiratory tract infections in adults, particularly in the setting of chronic bronchitis and emphysema (refs. 3, 4, 5, 6, 7, 8 and 9).
M. catarrhalis
also causes sinusitis in children and adults (refs. 10. 11, 12, 13 and 14) and occasionally causes invasive disease (refs. 15, 16, 17, 18, 19 and 20). In the hospital setting,
M. catarrhalis
has been suspected in outbreaks of nosocomial infection (ref. 21).
The incidence of otitis media caused by
M. catarrhalis
is increasing. As ways of preventing otitis media caused by pneumococcus and non-typeable
H. influenzae
are developed, the relative importance of
M. catarrhalis
as a cause of otitis media can be expected to further increase. Also antibiotic resistance is becoming common in clinical isolates of
M. catarrhalis
(ref 22). Thus, prior to 1970 no &bgr;-lactamase producing
M. catarrhalis
strains had been reported, but since the mid seventies, &bgr;-lactamase expressing strains have been detected with ever increasing frequency among isolates. Recent surveys suggest that 75% of clinical isolates produce &bgr;-lactamase.
Iron-restriction is a general host defence mechanism against microbial pathogens, however, it is not necessarily growth rate-limiting for all pathogens (ref. 23). A number of bacterial species including
Neisseria meningitidis
(ref. 24), N. gonorrhoeae (ref. 25) and
Haemophilus influenzae
(ref. 26), expresses two outer membrane proteins which specifically bind human transferrin (refs. 27, 28). The expression of these proteins is regulated by the amount of iron in the growth environment. Unlike the receptors of other bacterial species, the
M. catarrhalis
receptors have a preferred affinity for iron-laden (i.e., ferri-) transferrin (ref. 29). The two
M. catarrhalis
transferrin receptors (TfR) have molecular masses of 115 kDa (TfR1) and about 80 to 90 kDA (TfR2) (ref. 27).
The outer membrane protein OMP B2 (refs. 30, 31) has a molecular mass similar to that of TfR2, and it has been reported that the expression of OMP B2 is iron-regulated (ref. 32).
Yu and Schryvers (ref. 29) describe a method for purification of the transferrin receptor proteins TfR1 and TfR2 from
M. catarrhalis
by selective elution from a transferrin-sepharose affinity column using the denaturing agent guanidine HCl.
In this method of the preparative isolation of receptor proteins, a suspension of iron-deficient crude
M. catarrhalis
membranes was solubilized by the addition of EDTA to 20 mM and Sarkosyl to 0.75% and then the mixture was centrifuged for 15 minutes at 20,000× g to remove debris. Twenty ml of Fe
2
hTf-Sepharose (i.e. an iron-saturated form of human transferrin) was added to the supernatant and then incubated with mixing at room temperature for 45 minutes. The mixture was applied to a column and after removing the binding solution, the resin was washed with an additional 250 ml of 50 mM Tris-HCl, 1 M NaCl, 20 mM EDTA, 0.75% Sarkosyl, pH 8 buffer containing 250 mM guanidine HCl. The TfR2 protein was eluted using a buffer (lacking Sarkosyl) containing 1.5 M guanidine-HCl and subsequently the TfR1 protein was eluted by the application of a buffer containing 4 M guanidine-HCl. The fractions were dialysed against 50 mM Tris HCl, pH 8 over a 24 hour period.
In a further modification of this procedure, apohTf-Sepharose was mixed with the solubilized membrane preparation in order to bind TfR1 and then the treated solubilized membrane preparation was exposed to Fe
2
hTf-Sepharose to bind the remaining TfR2. The affinity resins were subsequently washed and the receptor proteins eluted as described above.
M. catarrhalis
infection may lead to serious disease. It would be advantageous to provide non-denatured transferrin receptor protein from
M. catarrhalis
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 purified and isolated transferrin receptor protein of
Moraxella catarrhalis
and other Moraxella strains, having an apparent molecular mass of about 80 to 90 kDa.
In accordance with one aspect of the invention, there is provided an isolated and purified, non-denatured transferrin receptor protein of a Moraxella strain having an apparent molecular mass of about 80 to about 90 kDa, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), or a fragment or an analog thereof (TfR2). The transferrin receptor protein may be substantially in its native conformation (so as to have substantially retained the characteristic immunogenicity of the transferrin receptor protein in the Moraxella strain) and may be isolated from a
M. catarrhalis
strain, such as from
M. catarrhalis
4223, 5191 or 135. Such isolated and purified transferrin receptor protein of apparent molecular mass of about 80 to about 90 kDa is substantially free from non-80 to 90 kDa Moraxella proteins, particularly the OMP B2 protein and the transferrin receptor protein having an apparent molecular mass of about 105 kDa of the Moraxella strains, phospholipids and lipopolysaccharide of Moraxella. The about 80 to about 90 kDa transferrin receptor 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.
The present invention also provides an immunogenic composition comprising an immunoeffective amount of an active component, which may be the transferrin receptor protein or fragment or analog thereof, provided herein along with a pharmaceutically acceptable carrier therefor. The immunogenic composition may be formulated as a vaccine for in vivo administration to a host to confer protection against diseases caused by a strain of Moraxella, particularly
M. catarrhalis.
The immunogenic composition may be formulated as a microparticle capsule, ISCOM or liposome preparation. The immunogenic composition may be used in combination with a targeting

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