Lactoferrin receptor protein

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Conjugate or complex

Reexamination Certificate

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C424S203100, C424S249100, C424S250100, C424S251100, C530S350000

Reexamination Certificate

active

06344200

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the field of immunology and is particularly concerned with a novel lactoferrin receptor protein isolated and purified from bacterial pathogens and methods of production, and uses thereof.
BACKGROUND TO THE INVENTION
Most living organisms require a continuous supply of iron to maintain growth and thus have evolved efficient mechanisms for acquisition of iron under conditions of limitation (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). A common mechanism found in many bacterial species involves the synthesis and secretion of small iron-chelating molecules, siderophores, which complex with iron and are subsequently bound and internalized via specific receptors at the bacterial surface (ref. 2). This mechanism is effective in a wide variety of environments and is often found in bacterial species that are present in a variety of ecological niches.
The vertebrate host provides an iron-restricted environment to potential bacterial pathogens, largely due to the sequestration of extracellular iron by the host iron-binding glycoproteins, namely transferrin (Tf) and lactoferrin (Lf). Although a siderophore-mediated mechanism should be effective in this setting, some bacterial species have evolved alternative mechanisms of iron acquisition that are adapted to their particular host. Thus, some members of the Pasteurellaceae and Neisseriaceae are capable of specifically binding and effectively acquiring iron from the host Tf and in some cases, Lf. This is mediated by receptors present at the bacterial surface whose expression is induced by restricting the level of available iron in the medium.
Receptors that are specific for Tf from the host (ref. 3) have been identified in a variety of important human and veterinary pathogens as well as some commensal species (Table 1). To date, Tf receptors have only been identified in bacterial species within the Pasteurellaceae and Neisseriaceae. In most species the Tf receptor has been shown to consist of two proteins, Tf-binding protein I (Tbp1) and Tf-binding protein 2 (Tbp2). The genes encoding these proteins have been cloned from
Neisseria meningitidis
(ref. 13),
N. gonorrhoeae
(refs. 14 and 15),
Haemophilus influenzae
(ref. 16) and
Actinobacillus pleuropneumoniae
(refs. 17 and 18). The predicted amino acid sequences of Tbp2 proteins reveal homology to the lipoprotein signal peptidase recognition site suggesting that it is lipid-modified and retains an association with the outer membrane via its lipid tail. Lipidation of Tbp2 has been confirmed by labelling (refs. 13 and 19) and evidence is accumulating that Tbp2 is largely surface exposed (refs. 20 and 21). Isogenic mutants deficient in the production of Tbp2 demonstrate severely limited ability to utilize transferrin as a sole iron source during in vitro growth studies, suggesting Tbp2 plays an important, albeit not essential role in iron acquisition (refs. 14, 16, 22).
Based on its homology with other TonB-dependant outer membrane proteins Tbp1 receptor proteins are believed to have several regions which span the outer membrane (ref. 23) (see ref. 35 for a topological model that can be applied to Tbp1). Similarly, based on the results obtained with the FepA receptor (ref. 25), Tbp1 is thought to act as a gated pore which allows the passage of iron from the transferrin and lactoferrin molecules, which are themselves not internalized, to the periplasm where it is bound by the ferric iron-binding protein, FbpA. Two additional proteins FbpB and FbpC are believed to be involved in the transport of iron across the cytoplasmic membrane.
The transport of iron across the outer membrane is believed to occur in a TonB-dependent manner, in that amino acid sequences referred as the “TonB box”, located in a number of TonB-dependent outer membrane receptor proteins have also, been identified in Tbp1. The inability to utilize human transferrin following insertional inactivation of the
H. influenzae
TonB homologue clearly supports this theory. In addition, mutants in which the Tbp1 protein has been insertionally inactivated are unable to utilize transferrin as a sole iron source, supporting its essential role in iron acquisition from transferrin.
Bacterial lactoferrin receptors have only been described for human pathogens in the Neisseriaceae, and were thought to consist of a single protein, Lbp1. Amino acid sequence analysis of the Lbp1protein shows high homology to Tbp1 (refs. 26, 35, 36), and isogenic mutant analysis deficient in Lbp1suggests an essential role of Lbp1in iron acquisition (refs. 24 and 28). Recent genetic evidence suggests that similar to the tbpBA operonic organization, an open reading frame is located immediately upstream of the lbpA structural gene which may encode a Tbp2 homologue, Lbp2(ref. 35).
Properties of lactoferrin receptor proteins of bacterial pathogens indicate that these proteins have utility in diagnosis of and vaccination against diseases caused by such bacterial pathogens that produce lactoferrin receptor proteins or proteins capable of raising antibodies specifically reactive with lactoferrin receptor proteins.
It would be advantageous to provide purified lactoferrin receptor proteins (and methods of purification thereof) for use as antigens, 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 lactoferrin receptor protein from a bacterial pathogen having a molecular mass of about 70,000 and about 90,000 daltons wherein the molecular mass is determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
In accordance with one aspect of the invention, there is provided a lactoferrin receptor protein isolated and purified from a bacterial pathogen and having an apparent molecular mass of about 70,000 to about 90,000 daltons as determined by SDS-PAGE.
The bacterial pathogens from which the lactoferrin receptor protein may be isolated and purified include
Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella catarrhalis, Moraxella bovis
and
Moraxella lacunata.
The about 70 to about 90 kDa lactoferrin 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 lactoferrin receptor protein, sometimes referred to herein by the designation “Lbp2”, may be provided in a form substantially free from a lactoferrin receptor protein having a molecular weight of about 100,000 to about 105,000 daltons, as determined by SDS-PAGE, sometimes referred to herein by the designation “Lbp1”.
Alternatively, the novel lactoferrin receptor protein provided herein may comprise a mixture of the lactoferrin receptor proteins. In another aspect of the present invention, therefore, there is provided a composition comprising a first lactoferrin receptor protein (Lbp1) having a molecular weight of about 100,000 to about 105,000 and a second lactoferrin receptor protein having a molecular weight of between about 70,000 and about 90,000 daltons, wherein the molecular weights are determined by SDS-PAGE and the lactoferrin receptor proteins are isolated and purified from a bacterial pathogen, which may be any of the pathogens mentioned above.
Such composition may be at least about 70% pure by weight, preferably at least about 90% pure by weight. Such composition also may be provided substantially free from transferrin receptor proteins of the pathogen.
The present invention also provides an immunogenic composition comprising an immunoeffective amount of an active component, which may be the novel lactoferrin rec

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