| Title of Invention | INTERACTION OF MORAXELLA CATARRHALIS WITH EPITHELIAL CELLS, EXTRACELLUAR MATRIX PROTEINS AND THE COMPLEMENT SYSTEM |
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| Abstract | The present invention relates to extra cellular matrix proteins of Moraxella catarrhalis and their ability to interact with epithelial cells via cellassociated fibronectin and laminin, and also to their ability to inhibit the complement system. These extracellular proteins are useful in the preparation of vaccines. The present invention provides peptides interacting with the fibronectin, laminin and complement system. |
| Full Text | INTERACTION OF MORAXELLA CATARRHALIS WITH EPITHELIAL CELLS, EXTRACELLULAR MATRIX PROTEINS AND THE COMPLEMENT SYSTEM Technical field of the invention The present invention relates to Moraxella catarrhalis and their ability to interact with epithelial cells via extracellular matrix proteins such as fibronectin and laminin, and also to their ability to inhibit the complement system. The interaction with these extracellular proteins is useful in the preparation of vaccines. Background art The ability to bind epithelial cells is of great importance for several bacterial species. For example, Staphylococcus aureus and Streptococcus pyogenes possess fibronectin binding proteins (FnBP) with related sequence organization. These FnBP are known as Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs). They exploit the modular structure of fibronectin forming extended tandem beta-zippers in its.binding to fibronectin. [27:, 39, 47, 73] The function is to mediate bacterial adhesion and invasion of host cells. The important mucosal pathogen Moraxella catarrhalis is the third leading bacterial cause of acute otitis media in children after -Streptococcus pneumoniae and Haemophilus influenzae.[14, 40, 55] M. catarrhalis is also one of the most common inhabitants of the pharynx of healthy children. Furthermore, M. catarrhalis is also a common cause of sinusitis and lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). [74] The success of this species in patients with COPD is probably related in part- to its large repertoire of adhesins. Recent years focus of research has been on the outer membrane proteins and their interactions with the human host. [6, 48, 56] Some of these outer membrane proteins appear to have adhesive functions including amongst others, M. catarrhalis IgD binding protein (MID, also designated Hag), protein CD, M. catarrhalis adherence protein (McaP) and the ubiquitous surface proteins (Usp).[1, 22, 33, 48, 61, 81, 84] Summary of the invention In view of the fact that M. catarrhalis has been found to be such a leading cause of infections in the upper and lower airways, there is a current need to develop vaccines which can be used against M. catarrhalis. The aim of the present invention has therefore been to find out in which way M. catarrhalis interacts with epithelial cells in the body and affects the immune system. In this way, substances that can act as vaccines against M. catarrhalis can be developed. In this study, using M. catarrhalis mutants derived from clinical isolates, the inventors have been able to show that both UspAl and A2 bind fibronectin and laminin. Furthermore, the inventors have been able to show that M. catarrhalis interfere with the classical pathway of the complement system, and also to elucidate in which way they interfere. Many bacteria adhere to epithelial cells via fibronectin binding MSCRAMMS.[54, 77] Pseudomonas aeruginosa has a FnBP that binds to cellular associated fibronectin on nasal epithelial cells.[69] Blocking the bacteria-fibronectin protein interactions may help the host tissue to overcome the infection. In fact, it has been shown that antibodies against a S. aureus FnBP resulted in rapid clearance of the bacteria in infected mice.[71] Recombinant truncated UspAl/A2 proteins together with smaller fragments spanning the entire molecule have been tested according to the present invention for fibronectin binding. Both UspAl and A2 bound fibronectin and the fibronectin binding domains were found to be located within UspAl299"452 and UspA2165"318. These two truncated proteins both inhibited binding of M. catarrhalis to Chang conjunctival epithelial cells to a similar extent as anti-fibronectin antibodies. The observations made show that both M. catarrhalis UspAl and A2 are involved in the adherence to epithelial cells via cell-associated fibronectin. The biologically active sites within UspAl299'452 and UspA2165"318 are therefore suggested as potential candidates to be included in a vaccine against M. catarrhalis. Further, the inventors have studied and characterized binding of M. catarrhalis to laminin. M. catarrhalis is a common cause of infectious exacerbations in patients with COPD. The success of this species in patients with COPD is probably related in part to its large repertoire of adhesins. In addition, there are pathological changes such as loss of epithelial integrity with exposure of basement membrane where the laminin layer itself is thickened in smokers.[4] Some pathogens have been shown to be able to bind laminin and this may contribute to their ability to adhere to such damaged and denuded mucosal surfaces. These include pathogens known to cause significant disease in the airways such as S. aureus and P. aeruginosa amongst others.[7, 63] The present inventors have been able to show that M. catarrhalis ubiquitous surface protein (Usp) Al and A2 also bind to laminin. Laminin binding domains of UspAl and A2 were, amongst others, found within the ^-terminal halves of UspAl50"491 and UspA230"351. These domains are also containing the fibronectin binding domains. However, the smallest fragments that bound fibronectin, UspAl299"452 and UspA2165"318, did not bind laminin to any appreciable extent. Fragments smaller than the N-terminal half of UspAl (UspAl50 491) lose all its laminin binding ability, whereas with UspA2, only UspA230"170 bound laminin albeit at a lower level than the whole recombinant protein (UspA230"539) . These findings suggest that different parts of the molecule might have different functional roles. UspAl50"770 was also found to have laminin binding properties. Comparing the smallest laminin binding regions of UspAl and A2, we find that there is, however, little similarity by way of amino acid homology between UspA230"170 and UspAl50"491 (data not shown). This is not surprising as it is a known fact that both proteins have a 'lollipop!-shaped globular head structure despite having only 22% identity in both N terminal halves.[2, 32] The biologically active sites within UspAl50"770 and UspA230"539 are suggested as potential candidates to be included in a vaccine against M. catarrhalis. Finally, the inventors have studied the interaction between M. catarrhalis ubiquitous surface proteins Al and A2 and the innate immune system, and have found that M. catarrhalis interferes with the complement system. The complement system is one of the first lines of innate defence against pathogenic microorganisms, and activation of this system leads to a cascade of protein deposition on the bacterial surface resulting in formation of the membrane attack complex or opsonization of the pathogen followed by phagocytosis. [85, 86] One of the most important complement proteins is C3, which is present in the circulation in a concentration similar to some immunoglobulins (1-1.2 nig/ml). C3 does not only play a crucial role as an opsonin, but also is the common link between the classical, lectin and alternative pathways of the complement activation. The alternative pathway functions as amplification loop for the classical and lectin pathways and can also be spontaneously activated by covalent attachment of C3 to the surface of a microbe in the absence of complement inhibitors. C3 deposition requires the presence of an internal thioester bond, formed in the native protein by the proximity of a sulfhydryl group (Cys1010) and a glutamyl carbonyl (Gin1012) on the C3 Several functions have been attributed to the UspA family of proteins. UspAl expression is essential for the attachment of M. catarrhalis to Chang conjunctival epithe- lial cells and Hep-2 laryngeal epithelial cells. [43, 49] In a more recent study, UspAl was shown to bind carcinoem-bryonic antigen related cell adhesion molecules (CEACAM) expressed in the lung epithelial cell line A549.[31] Purified UspAl has also been shown to bind fibronectin in dot blot experiments while purified UspA2 did not. [49] Both UspAl and A2 may play important roles for M. catarrhalis serum resistance.[1, 5, 58, 60] The present invention demonstrates that both UspAl and A2 are determinants for Af. catarrhalis binding to fibronectin and laminin in the clinical isolates M. catarrhalis BBH18 and RH4. Interestingly, recombinant UspAl and A2 derived from M. catarrhalis Bc5 both bound fibronectin to the same extent. The binding domains for fibronectin were found within amino acid residues 299 to 452 of UspAl and 165 to 318 of UspA2. These two domains share 31 amino acid residues sequence identity. Importantly, truncated protein fragments containing these residues in UspAl and UspA2 were able to inhibit M. catarrhalis binding to Chang epithelial cells suggesting that the interactions with these cells were via cell-associated fibronectin. The binding domains for laminin were found within the amino acid residues mentioned above. Binding assays with recombinant proteins revealed that the major binding regions were localized in the N-terminal parts, where both proteins form a globular head. Bacterial factors mediating adherence to tissue and extracellular matrix (ECM) components are grouped together in a single family named ^microbial surface components recognizing adhesive matrix molecules" (MSCRAMMS). Since UspAl/A2both bind fibronectin and laminin, these proteins can be designated MSCRAMMS. According to one aspect the present invention provides a peptide having sequence ID no. 1, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to another aspect the present invention provides a peptide having sequence ID no. 2, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation/' sulphonation or glycosylation products and other secondary processing products thereof. According to a further aspect the present invention provides a peptide having sequence ID no. 3, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to another aspect the present invention provides a peptide having sequence ID no. 4, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to a further aspect the present invention provides a peptide having sequence ID no. 5, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to a further aspect the present invention provides a peptide having sequence ID no. 6, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to another aspect the present invention provides a peptide having sequence ID no. 7, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to another aspect the present invention provides a peptide having sequence ID no. 8, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to another aspect the present invention provides a peptide having sequence ID no. 9, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to another aspect the present invention provides a peptide having'sequence ID no. 10, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. According to another aspect, the present invention provides use of at least one peptide according to the invention for the production of a medicament for the treatment or prophylaxis of an infection, preferably an infection caused by itf. catarrhalis, in particular caused by carriage of M, catarrhalis on mucosal surfaces. According to another aspect, the invention further provides a ligand comprising a fibronectin binding domain, said ligand consisting of an amino acid sequence selected from the group consisting of Sequence ID No. 1, Sequence ID No. 2 and Sequence ID No. 3, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. The invention further provides a ligand comprising a laminin binding domain, said ligand consisting of an amino acid sequence selected from the group consisting of Sequence ID No. 4 to Sequence ID No. 8, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. Further, the present invention provides a ligand comprising a C3 or C3met binding domain, said ligand consisting of an amino acid sequence selected from the group consisting of Sequence ID No. 4, Sequence ID No. 6, Sequence ID No. 9 and Sequence ID No. 10, and fragments, homologues, functional equivalents, derivatives, degenerate or hydroxylation, sulphonation or glycosylation products and other secondary processing products thereof. Further, the present invention provides a medicament comprising one or more ligands according to the invention and one or more pharmaceutically acceptable adjuvants, vehicles, excipients, binders, carriers, or preservatives. The present invention further provides a vaccine comprising one or more ligands according to the present invention and one or more pharmaceutically acceptable adjuvants, vehicles, excipients, binders, carriers, or preservatives. The present invention also provides a method of treating or preventing an infection in an individual, preferably an infection caused by Af. catarrhalisf in particular caused by carriage of M. catarrhalis on mucosal surfaces, comprising administering a pharmaceutically effective amount of a medicament or vaccine according to the present invention. Finally, the present invention also provides a nucleic acid sequence encoding a ligand, protein or peptide of the present invention, as well as homologues, polymorphisms, degenerates and splice variants thereof-Further disclosure of the objects, problems, solutions and features of the present invention will be apparent from the following detailed description of the invention with reference to the drawings and the appended claims. The expression ligand as it is used herein is intended to denote both the whole molecule which binds to the receptor and any part thereof which includes the receptor binding domain such that it retains the receptor binding property. Ligands comprising equivalent receptor binding domains are also included in the present invention. The expressions fragment, homologue, functional equivalent and derivative relate to variants, modifications and/or parts of the peptides and protein fragments according to the invention which retain the desired fibronectin, laminin, C3 or C3met binding properties. A homologue of UspAl according to the present invention is defined as a sequence having at least 72% sequence identity, as can be seen from table 1 below. A fragment according to the present invention is defined as any of the homologue sequences which are truncated or extended by 1, 2, 5, 10, 15, 20 amino acids at the N-terminus and/or truncated or extended by 1, 2, 5, 10, 15, 20 amino acids at the C-terminus. The expressions degenerate, hydroxylation, sulphonation and glycosylation products or other secondary processing products relate to variants and/or modifications of the peptides and protein fragments according to the invention which have been altered compared to the original peptide or protein fragment by degeneration, hydroxylation, sulphonation or glycosylation but which retain the desired fibronectin, laminin, C3 or C3met binding properties. The present invention concerns especially infections caused by Moraxella catarrhalis. A peptide according to the present invention can be used for the treatment or prophylaxis of otitis media, sinusitis or lower respiratory tract infections. Accordingly, the present invention provides a ligand isolated from Moraxella catarrhalis outer membrane protein which has laminin and/or fibronectin and/or C3-binding, wherein said ligand is a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10 which are derived from the full-length Moraxella catarrhalis BC5 UspAl & UspA2 sequences shown below, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. Full-length UspAl from Moraxella catarrhalis strain BC5: MNKIYKVKKN AAGHLVACSE FAKGHTKKAV LGSLLIVGIL GMATTASAQK VGKATNKISG GDNNTANGTY LTIGGGDYNK TKGRYSTIGG GLFNEATNEY STIGSGGYNK AKGRYSTIGG GGYNEATNQY STIGGGDNNT AKGRYSTIGG GGYNEATIEN STVGGGGYNQ AKGRNSTVAG GYNNEATGTD STIAGGRKNQ ATGKGSFAAG IDNKANADNA VALGNKNTIE GENSVAIGSN NTVKKGQQNV FILGSNTDTT NAQNGSVLLG HNTAGKAATI VNSAEVGGLS LTGFAGASKT GNGTVSVGKK GKERQIVHVG AGEISDTSTD AVNGSQLHVL ATVVAQNKAD IKDLDDEVGL LGEEINSLEG EIFNNQDAIA KNQADIKTLE SNVEEGLLDL SGRLLDQKAD IDNNINNIYE LAQQQDQHSS DIKTLKNNVE EGLLDLSGRL IDQKADLTKD IKALESNVEE GLLDLSGRLI DQKADIAKNQ ADIAQNQTDI QDLAAYNELQ DAYAKQQTEA IDALNKASSA NTDRIATAEL GIAENKKDAQ IAKAQANENK DGIAKNQADI QLHDKKITNL GILHSMVARA VGNNTQGVAT NKADIAKNQA DIANNIKNIY ELAQQQDQHS SDIKTLAKVS AANTDRIAKN KAEADASFET LTKNQNTLIE QGEALVEQNK AINQELEGFA AHADVQDKQI LQNQADITTN KTAIEQNINR TVANGFEIEK NKAGIATNKQ ELILQNDRLN RINETNNHQD QKIDQLGYAL KEQGQHFNNR ISAVERQTAG GIANAIAIAT LPSPSRAGEH HVLFGSGYHN GQAAVSLGAA GLSDTGKSTY KIGLSWSDAG GLSGGVGGSY RWK Full-length UspA2 from Moraxella catarrhalis strain BC5: MKTMKLLPLK IAVTSAMIIG LGAASTANAQ AKNDITLEDL PYLIKKIDQN ELEADIGDIT ALEKYLALSQ YGNILALEEL NKALEELDED VGWNQNDIAN LEDDVETLTK NQNAFAEQGE AIKEDLQGLA DFVEGQEGKI LQNETSIKKN TQKNLVNGFE IEKNKDAIAK NNESIEDLYD FGHEVAESIG EIHAHNEAQN ETLKGLITNS IENTNNITKN KADIQALENN WEELFNLSG RLIDQKADID NNINNIYELA QQQDQHSSDI KTLKKNVEEG LLELSDHIID QKTDIAQNQA NIQDLATYNE LQDQYAQKQT EAIDALNKAS SENTQNIEDL AAYNELQDAY AKQQTEAIDA LNKASSENTQ NIEDLAAYNE LQDAYAKQQA EAIDALNKAS SENTQNIAKN QADIANNITN IYELAQQQDK HRSDIKTLAK TSAANTDRIA KNKADDDASF ETLTKNQNTL IEKDKEHDKL ITANKTAIDA NKASADTKFA ATADAFTKNG NAITKNAKSI TDLGTKVDGF DSRVTALDTK VNAFDGRITA LDSKVENGMA AQAALSGLFQ PYSVGKFNAT AALGGYGSKS AVAIGAGYRV NPNLAFKAGA AINTSGNKKG SYNIGVNYEF In a preferred embodiment, the ligand is a polypeptide [or polypeptide truncate compared with a wild-type polypeptide] comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. The term ligand is used herein to denote both the whole molecule which binds to laminin and/or fibronectin and/or C3 and any part thereof which includes a laminin and/or fibronectin and/or C3-binding domain such that it retains the. respective binding property. Thus "ligand" encompasses molecules which consist only of the laminin and/or fibronectin and/or C3-binding domain i.e. the peptide region or regions required for binding. For the purposes of this invention laminin, fibronectin or C3-binding properties of a polypeptide can be ascertained as follows: For, the purposes of this invention laminin, fibronectin or C3-binding properties of a polypeptide can be ascertained as follows: Polypeptides can be labelled with 125Iodine or other radioactive compounds and tested for binding in radio immunoassays (RIA) as fluid or solid phase (e.g., dot blots). Moreover, polypeptides can be analysed for binding with enzyme-linked immunosorbent assays (ELISA) or flow cytometry using appropriate antibodies and detection systems. Interactions between polypeptides and laminin, fibronectin, or C3 can further be examined by surface plasmon resonance (Biacore). Examples of methods are exemplified in detail in the Material and Methods section. In another preferred embodiment, the polypeptide [or polypeptide truncate compared with a wild-type polypeptide] comprises or consists of at least one of the conserved sequences from within SEQ ID NO: 1-10 which are identified in the alignment shown herein. Hence, in this embodiment, the polypeptide [or polypeptide truncate compared with a wild-type polypeptide] comprises of consists of at least one of: From UspAl (conserved fragments from the fibronectin binding domain - V separating alternative choices of an amino acid at a position) G T/V V S V G S/K Q/E/K/A G/N K/N/G/H/S E R Q I V N/H VGA G Q/N/E/K I S/R A/D T/D STDAVNGSQL H/Y ALA S/K/T T/A/V I/V STDAVNGSQL L L N/D L S G R L L/I DQKADIDNNIN N/H I Y E/D L A QQQDQHSSDIKTLK DQKADIDNNIN LAQQQDQHSSDIKTLK From UspA2 (conserved fragments from the fibronectin binding domain - V separating alternative choices of an amino acid at a position) KADIDNNIN N/H IYELAQQQDQHSSD I K/Q T/A L K/E K/N/S N V/I E/V E G/E L L/F E/N L S D/G H/R I/L I D Q K T/A D I/L A/T Q/K N/D From UspA2 (conserved fragments from the C3-binding domain - V separating alternative choices of an amino acid at a position) I E/Q DLAAYNELQDAYAKQQ A/T EAI DALNKA SSENTQNIAKNQADIANNI T/N NIYELAQQQ D K/Q H R/S SDIKTLAK T/A S A A N T D/N R I DLAAYNELQDAYAKQQ EAIDALNKASSENTQNIAKNQADIANNI It will be understood that the polypeptide ligands of the invention can comprise a laminin and/or fibronectin and/or C3-binding domain of sequence recited herein which is modified by the addition or deletion of amino acid residues to or from the sequences recited herein at either or both the N or C termini, which modified peptides retain the ability to bind laminin and/or fibronectin and/or C3, respectively. Accordingly, the invention further provides a ligand comprising or consisting of a polypeptide in which 50, 40, 30, 20, 10, 5, 3 or 1 amino acid residues have been added to or deleted from an amino acid sequence recited herein at either or both the N or C termini, wherein said modified polypeptide retains the ability to bind laminin and/or fibronectin and/or C3; and/or elicit an immune response against the non-modified peptide. By extension it is meant lengthening the sequence using the context of the peptide from the full-length amino acid sequence from which it is derived. As regards fragments of the polypeptides of the invention, any size fragment may be used in the invention (based on the homologue sequences/conserved regions/functional domatins discussed herein) provided that the fragment retains the ability to bind laminin and/or fibronectin and/or C3. It may be desirable to isolate a minimal peptide which contains only those regions required for receptor binding. Polypeptide ligands according to the invention may be derived from known Moraxella catarrhalis UspAl or UspA2 proteins by truncation at either or both of the N- and C-termini. Truncates are not the full-length native UspAl or A2 molecules. Accordingly, the invention further provides a wild-type UspAl sequence lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160 etc to 298 amino acids from the N-terminus, and/or lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180, 200 etc to 450 amino acids from the C-terminus. Preferably, the truncate retains fibronectin binding function (optionally also laminin and/or C3-binding). Accordingly the invention further provides a wild-type UspA2 sequence lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 164 amino acids from the N-terminus, and/or lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 180, 200 etc to 312 amino acids from the 'C-terminus. Preferably, the truncate retains fibronectin binding function (optionally also laminin and/or C3-binding). Possible truncates may be selected from those shown in the following table, all of which are within the scope of the invention. Accordingly the invention further provides a wild-type UspA2 sequence lacking at least (or exactly) 5, 10, 15, 20, 25 or 29 amino acids from the N-terminus, and/or lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160, 180, 200 etc to 453 amino acids from the O terminus. Preferably, the truncate retains laminin binding function (optionally also fibronectin and/or C3-binding). Possible truncates may be selected from those shown in the following table, all of which are within the scope of the invention. Accordingly the invention further provides a wild-type UspA2 sequence lacking (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160 etc. to 301 amino acids from the N-terminus, and/or lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160 or 172 amino acids from the C-terminus. Preferably, the truncate retains C3 binding function (optionally also fibronectin and/or laminin binding). Possible truncates may be selected from those shown in the following table, all of which are within the scope of the invention. Table 6. Possible combinations of truncations to the N- and C- termini of wild-type UspA2 protein Known wild-type UspAl sequences that may be truncated in this way are those of strains ATCC25238 (MX2; GenBank accession no. AAD43465), P44 (AAN84895), 035E (AAB96359), TTA37 (AAF40122), 012E (AAF40118), 046E (AAF36416), V1171 (AAD43469), TTA24 (AAD43467) (see Table 1/Figure 19); or BC5 (see above). Known wild-type UspA2 sequences that may be truncated in this way are those of strains 035E (GenBank accession no. O4407), MC317 (GenBank accession no. Q58XP4), E22 (GenBank accession no. Q848S1), V1122 (GenBank accession no, Q848S2), P44 (GenBank accession no. Q8GH86), TTA37 (GenBank accession no. Q9L961), 046E (GenBank accession no. Q9L962), 012E (GenBank accession no. Q9L963), V1171 (GenBank accession no. Q9XD51), TTA24 (GenBank accession no. Q9XD53), SP12-5 (GenBank accession no. Q8RTB2), ATCC25238 (GenBank accession no. Q9XD55) (see Table 2/Figure 20); or BC5 [Forsgren_UspA2] (see above). Ideally the UspAl or UspA2 truncate of this embodiment comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-10 or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof; or comprises or consists of at least one of the conserved sequences from within these regions which are identified in the alignment shown in herein, for example: From UspAl (conserved fragments from the fibronectin binding domain - V separating alternative choices of an amino acid at a position) G T/V V S V G S/K Q/E/K/A G/N K/N/G/H/S E R Q I V N/H VGA G Q/N/E/K I S/R A/D T/D STDAVNGSQL H/Y ALA S/K/T T/A/V I/V STDAVNGSQL L L N/D L S G R L L/I DQKADIDNNIN N/H I Y E/D L A QQQDQHSSDIKTLK DQKADI DNNIN LAQQQDQHSSDIKTLK From UspA2 (conserved fragments from the fibronectin binding domain - V separating alternative choices of an amino acid at a position) KADIDNNIN N/H IYELAQQQDQHSSD I K/Q T/A L K/E K/N/S N V/I E/V E G/E L L/F E/N L S D/G H/R I/L I D Q K T/A D I/L A/T Q/K N/D From UspA2 (conserved fragments from the C3-binding domain - V separating alternative choices of an amino acid at a position) I E/Q DLAAYNELQDAYAKQQ A/T EAI DALNKA SSENTQNIAKNQADIANNI T/N NIYELAQQQ D K/Q H R/S SDIKTLAK T/A S A A N T D/N R I DLAAYNELQDAYAKQQ EAI DALNKASSENTQNIAKNQADIANNI It may be convenient to produce fusion proteins containing polypeptide ligands as described herein. Accordingly, in a further embodiment, the invention provides fusion proteins comprising polypeptide ligands according to the invention. Preferably a fusion protein according to this embodiment is less than 50% identical to any known fully length sequence over its entire length. Such fusions can constitute a derivative of the polypeptides of the invention. Further derivatives can be the use of the polypeptides of the invention to as a carrier to covalently couple peptide or saccharide moieties. They may be coupled for instance to pneumococcal capsular oligosaccharides or polysaccharides, or Moraxella catarrhalis lipooligo-saccaharides, or non-typeable Haemophilus influenzae lipooligosaccaharides. Homologous peptides of the invention may be identified by sequence comparison. Homologous peptides are preferably at least 60% identical, more preferably at least 70%, 80%, 90%, 95% or 99% identical in ascending order of preference to the peptide sequence disclosed herein or fragments thereof or truncates of the invention over their entire length. Preferably the homologous peptide retains the ability to bind fibronectin and/or laminin and/or C3; and/or elicit an immune response against the peptide sequences disclosed herein or fragment thereof. Figures 19 and 20 show an alignment of peptide sequences of UspAl and UspA2 of different origin which indicates regions of sequence that are capable of being modified to form homologous sequences whilst retained function (i.e. fibronectin and/or laminin and/or C3 binding ability). Homologous peptides to the BC5 SEQ ID NO: 1-10 peptides are for instance those sequences corresponding to the BC5 sequence from other strains in Figures 19 and 20. Vaccines of the Invention The polypeptides /peptides /functional domains /homologues /fragments /truncates /derivatives of the invention should ideally be formulated as a vaccine comprising an effective amount of said component(s) and a pharmaceutically acceptable excipient. The vaccines of the invention can be used for administration to a patient for the prevention or treatment of Moraxella catarrhalis infection or otitis media or sinusitis or lower respiratory tract infections. They may be administered in any known way, including intramuscularly, parenternally, mucosally and intranasally. Combination Vaccines of the Invention The vaccines of the present invention may be combined with other Moraxella catarrhalis antigens for prevention or treatment of the aforementioned diseases. The present inventors have found in particular that Moraxella catarrhalis has at least 2 means of hampering the host immune system from attacking the organism. In addition to the interaction with C3 (and C4BP) mentioned in the Examples below, M. catarrhalis has a strong affinity for soluble and membrane bound human IgD through protein MID (also known as OMP106). Moraxella-dependent IgD-binding to B lymphocytes results in a polyclonal immunoglobulin synthesis which may prohibit production of specific monoclonal anti-moraxella antibodies. The fact that M. catarrhalis hampers the human immune system in several ways might explain why M. catarrhalis is such a common inhabitant of the respiratory tract. The inventors believe that the combination of antigens involved in the IgD-binding function (MID) and C3-binding function (UspAl and/or UspA2) can provide an immunogenic composition giving the host enhanced defensive capabilities against Moraxella's hampering of the human immune system thus providing an enhanced decrease in M. catarrhalis carriage on mucosal surfaces. A further aspect of the invention is therefore a vaccine composition comprising an effective amount of UspAl anci/or UspA2 (particularly the latter) (for instance full-length polypeptides or polypeptides /peptides /functional domains /homologues /fragments /truncates /derivatives of the invention as described herein, preferably which retains a C3-binding function) in combination with an effective amount of protein MID (for instance full-length polypeptides or polypeptides /peptides /functional domains /homologues /fragments /truncates /derivatives thereof, preferably which retain a human IgD-binding function), and a pharmaceutically acceptable excipient. Protein MID, and IgD-binding homologous/fragments/truncates thereof is described in WO 03/004651 (incorporated by reference herein). Particularly suitable fragments for this purpose is a polypeptide comprising (or consisting of) the F2 fragment described in WO 03/004651, or sequences with at least 60, 70, 80, 90, 95, 99% identity thereto which preferably retain human IgD-binding activity. The MID and UspA components of this combination vaccine may be separate from each other, or may be conveniently fused together by known molecular biology techniques. Brief description of the drawings Figure 1 shows thirteen M. catarrhalis strains tested for fibronectin binding (A). Strong fibronectin binding correlated to UspAl/A2 expression as detected by anti-UspAl/A2 pAb (B-I)- Flow cytometry profiles of M. catarrhalis BBH18 wild type and UspAl/A2 deficient mutants show an UspAl/A2-dependent binding to soluble fibronectin. The profiles of wild type clinical isolate (B and F) and corresponding mutants devoid of UspAl (C and G), or UspA2 (D and H) , and double mutants .(E and I) lacking both UspAl and UspA2 are shown- Bacteria were incubated with rabbit anti-UspAl/A2 or fibronectin followed by an anti-fibronectin pAb. FITC-conjugated rabbit pAb was subsequently added followed by flow cytometry analysis. A typical experiment out of three with the mean fluorescence intensity (MFI) for each profile is shown. Figure 2 shows that M. aatarrhalis RH4 UspA2 deficient mutants do not bind 125I-labeled fibronectin. E. coli BL21 was included as a negative control not binding fibronectin. Bacteria were incubated with 125I-labeled fibronectin followed by several washes and analyzed in a gamma counter. Fibronectin binding to the RH4 wild type expressing both UspAl and A2 was set as 100 %. The mean values of three independent experiments are shown. Error bars represent standard deviations (SD). Similar results were obtained with M. catarrhalis BBH18. Figure 3 shows pictures that verify that Af. catarrhalis mutants devoid of UspAl and UspA2 do not bind to immobilized fibronectin. M. catarrhalis wild type was able to adhere at a high density on fibronectin coated glass slides (A) . M. catarrhalis buspAl mutant was also retained at a high density (B), whereas M. catarrhalis AuspA2 and &uspAl/A2 double mutants adhered poorly (C and D) . Glass slides were coated with fibronectin and incubated with M. catarrhalis RH4 and its corresponding UspAl/A2 mutants. After several washes, bacteria were Gram stained. Figure 4 is a graph showing that recombinant UspAl and A2 bind to fibronectin in a dose-dependent manner. Specific fibronectin binding is shown for UspAl50"770 and UspA230"539. Both UspA proteins (40 nM) were coated on microtiter plates and incubated with increasing concentrations of fibronectin followed by detection with rabbit anti-human fibronectin pAb and HRP-conjugated anti-rabbit pAb. Mean values of three separate experiments are shown and error bars indicate SD. Figure 5. The active fibronectin binding domains for UspAl and UspA2 are located between amino acids 299 to 452 and 165 to 318, respectively. Truncated proteins derived from UspAl {A) and UspA2 {B) are shown. All fragments were tested for fibronectin binding in ELISA. Forty nM of each truncated fragment was coated on microtiter plates and incubated with 80 pg/ml and 120 pg/ml fibronectin for UspAl and UspA2, respectively. Bound fibronectin was detected with rabbit anti-fibronectin pAb followed by HRP-conjugated anti-rabbit pAb. Results are representative for three sets of experiments. Error bars represent SD. Figure 6 shows the sequence according to sequence ID No. 1, and the sequence homology between UspAl299"452 and UspA2165"318. The 31 identical amino acid residues are within brackets. Figure 7 shows that truncated UspAl50"491 and UspAl299"452 fragments competitively inhibit M. catarrhalis UspA-dependent fibronectin binding. M, catarrhalis &uspAl/A2 double mutants, which do not bind fibronectin, were included as negative qontrols. UspAl recombinant proteins were pre-incubated with 2 mg/100 ml fibronectin before incubation with M. catarrhalis. The mean fluorescence values (MFI) of M. catarrhalis with bound fibronectin detected by FITC conjugated anti-fibronectin pAb in flow cytometry are shown. UspAl50"491 and UspAl299"452 resulted in 95 % and 63 % inhibition respectively. Error bars represent mean ± SD of three independent experiments. Figure 8 shows that UspAl299"452 and UspA2165"318 inhibit M. catarrhalis adherence to Chang conjunctival cells via cell-associated fibronectin. Chang epithelial cells expressed fibronectin on the surface as revealed by an anti-fibronectin pAb and flow cytometry (A). Pre-incubation with the fibronectin binding proteins UspAl299"452, UspA2165"318, or anti-fibronectin pAb resulted in significantly reduced binding by M. catarrhalis RH4 as compared to control recombinant proteins (UspAl433"580 and UspA230"177) and a control antibody (anti-ICAMl mAb) (B). P paired Student's t test. Mean values of three separate experiments are shown and error bars indicate SD. Fig. 9A shows binding of M. catarrhalis RH4 to laminin via UspAl and A2. M. catarrhalis RH4 wild type {wt) strongly bound to immobilized laminin with a mean OD of 1.27. RMAuspAl showed mean OD of 1,14 (89.8 % of the wild type). RH4£uspA2 and the double mutant RH4AuspAl/A2 had a mean OD of 0.19 and 0.23 respectively (15.0% and 18.1% of the wild type). This was not significantly different from the residual adhesion to bovine serum albumin coated plates. Thirty pg/ml of laminin or bovine serum albumin were coated on microtiter plates. They were blocked followed by incubation with bacteria suspension and finally washed. Bound bacteria was detected with anti-MID pAb and HRP-conjugated anti-rabbit pAb. The mean results of 3 representative experiments are shown. Error bars represent standard deviations (SD). Fig. 9B shows the binding of recombinant UspAl and A2 laminin in a dose-dependent manner. Specific laminin binding is shown for UspAl50"770 and UspA230"539.Both UspA proteins (40 nM) were coated on microtiter plates and incubated with increasing concentrations of laminin followed by detection with rabbit anti-laminin pAb and HRP-conjugated anti-rabbit pAb. Mean values of three separate experiments are shown and error bars indicate SD. Fig. 10 A and B show that the active laminin binding domains for UspAl50"770 (A) and UspA230"539 (B) are located in the N-terminal halves. Forty nM of recombinant UspAl50"770 and UspA230"539 together with the truncated proteins were coated on microtiter plates and incubated with 20 pg/ml of laminin followed by detection with rabbit anti-laminin pAb and HRP-conjugated anti-rabbit pAb. Mean values of three separate experiments are shown and error bars indicate SD. Fig. 11 is a schematic illustration of C3, covalent bound C3b and C3met. (A) The C3-molecule in serum consists of one a-chain and one (3-chain. (B) The a-chain contains an internal thioester site that after activation can attach covalently to a microbial surface. (C) The C3 has been treated with methylamine, which becomes covalently attached to the thioester. Fig, 12 illustrates that M. catarrhalis counteracts the classical and alternative pathways of the complement system by the outer membrane proteins UspAl and A2. (A) M. catarrhalis RH4 wild-type (wt), the kuspAl, the AuspA2 or the t\uspAl/A2 mutants were incubated in the presence of 10 % NHS. (B) The AuspAl/A2 mutant was incubated with 10 % NHS supplemented with either EDTA or Mg-EGTA. Bacteria were collected at the indicated time points. After overnight incubation, colony forming units (cfu) were counted. The number of bacteria at the initiation of the experiments was defined as 100 %. Mean values of three separate experiments are shown and error bars indicate S.D. (A) The mean values after 5 min for the buspAl, the kuspA2 or the &uspAl/A2 mutants were significantly different from the wild-type (P Fig. 13 illustrates that Moraxella catarrhalis binds C3 in serum independently of complement activation. Flow cytometry profiles showing C3 binding to (A) M, catarrhalis RH4 or (B) Streptococcus pneumoniae. Bacteria were incubated with NHS or NHS pretreated with EDTA. Thereafter, a rabbit anti-human C3d pAb and as a seconddary layer a FITC-conjugated goat anti-rabbit pAb were added followed by flow cytometry analysis. Bacteria in the absence of NHS, but in the presence of both pAb, were defined as background fluorescence. One representative experiment out of three is shown. Fig. 14 illustrates that M. catarrhalis non-covalently binds purified methylamine-treated C3 in a dose-dependent manner, and that the binding is based on ionic interactions. Flow cytometry profiles showing (A) binding with increasing concentrations of C3met. (B) The mean fluorescence intensity (mfi) of each profile in panel (A) is shown. (C) C3met binding of RH4 decreases with increasing concentrations of NaCl. Bacteria were incubated with C3met with or without NaCl as indicated. C3met binding was measured by flow cytometry as described in Figure 3. Error bars indicate SD. * P Z 0.05, ** P <. p> Fig. 15 illustrates that flow cytometry profiles of M. catarrhalis RH4 wild type and UspAl/A2 deficient mutants show a UspAl/UspA2-dependent C3met/ C3 binding. The profiles of a wild type clinical isolate (Af F, K) and corresponding mutants devoid of protein MID (B, G, L) , UspAl (C, H, M) , UspA2 (D, I, N), or both UspAl and UspA2 (E, J, 0) are shown. Bacteria were incubated with C3met (A-E), NHS-EDTA (F-J) or NHS (K-0) and detected as outlined in Figure 3, One typical experiment out of three with the mean fluorescence intensity (mfi) for each profile is shown. Fig. 16 illustrates that C3met binds to purified recombinant UspA230'539, whereas only a weak C3met binding to UspAl50"770 is observed. Furthermore, the C3met binding region of UspA2 was determined to be located between the amino acid residues 200 to 458. (A) The recombinant UspAl50'770 and UspA230"539 were immobilized on a nitrocellulose membrane. The membrane was incubated with [125I]-labelled C3met overnight and bound protein was visualized with a Personal FX (Bio-Rad) using intensifying screens. The recombinant protein MID962"1200 was included as a negative control. (B) UspAl50"770, UspA230"539 and a series of truncated UspA2 proteins were coated on microtiter plates and incubated with C3met, followed by incubation with goat anti-human C3 pAb and HRP-conjugated anti-goat pAb. The mean values out of three experiments are shown. The background binding was subtracted from all samples. Error bars correspond to S.D. * P £ 0.05/ ** P ^ 0.01, *** P £ 0.001. Fig. 17 illustrates that addition of recombinant UspAl50"770 and UspA230~539 to serum inhibit C3b deposition and killing of M. catarrhalis via the alternative pathway. Flow cytometry profiles show C3b-deposition on RH4AuspAl/A2 after incubation with (A) NHS or NHS preincubated with recombinant (rec.) UspAl50"770 and UspA230~539, or (B) NHS-Mg-EGTA or NHS-Mg-EGTA preincubated with UspAl50"770 and UspA230"539. After addition of the various NHS combinations, bacteria were analyzed as described in Figure 13. (C) RR4&uspAl/A2 was incubated with 10 % NHS or NHS-Mg-EGTA. For inhibition, the NHS-Mg-EGTA was incubated with 100 nM UspAl50"770 and/ or UspA230"539 before addition of bacteria. Bacteria were collected at the indicated time points. The number of bacteria at the initiation of the experiments was defined as 100 %. Mean values of three separate experiments are shown and error bars indicate S.D. The time points 10, 20 and 30 min for the kuspAl/A2 mutant preincubated with recombinant proteins were significantly different from the LuspAl/A2 mutant incubated with Mg-EGTA alone (P Fig. 18 illustrates that recombinant UspAl50"770 and UspA230"539 decrease haemolysis of rabbit erythrocytes by inhibition of the alternative pathway. NHS was incubated with or without 100 nM UspAl50"770 and/ or UspA230"539 at 37 °C for 30 min. NHS at the indicated concentrations was thereafter added to rabbit erythrocytes. After incubation for 30 min, the suspensions were centrifuged and the supernatants were measured by spectrophotometry. Maximum haemolysis in each experiment was defined as 100 %. Mean values of three separate experiments are shown and error bars correspond to S.D. The results obtained with NHS + UspA230"539 and NHS + UspAl50"770/ UspA230"539 at NHS concentrations of 2, 3 and 4% were significantly different from the NHS control (F Fig. 19 illustrates a pileup-analysis of UspAl for eight different strains, to show the homology of different parts of UspAl. Fig. 20 illustrates a pileup-analysis of UspA2 for thirteen different strains to show the homology of different parts of UspA2. Fig. 21 illustrates %identity in regions identified on Forsgren sequence computed as the ratio between the number of exact matches and the length of the region alignment, where the region alignment is that part of the above total alignment containing the Forsgren region. Materials and methods Interaction between M. catarrhalis and fibronectin Bacterial strains and culture conditions The sources of the clinical M. catarrhalis strains are listed in table 7. M. catarrhalis BBH18 and RH4 mutants were constructed as previously described.[23, 58] The M. catarrhalis strains were routinely cultured in brain heart infusion (BHI) liquid broth or on BHI agar plates at 37 °C. The UspAl-deficient mutants were cultured in BHI supplemented with 1.5 pg/ml chloramphenicol (Sigma, St, Louis, MO), and UspA2-deficient mutants were incubated with 7 pg/^1 zeocin (Invitrogen, Carlsbad, CA). Both chloramphenicol and zeocin were used for growth of the double mutants. Table 7. Clinical strains of M. catarrhalis used in the present study Strain Clinical Source Reference BBH18 Sputum [53] Dl Sputum [53] Ri49 Sputum [53] CIO Sputum [10] F16 Sputum [10] Bro2 Respiratory tract [53] Z14 Pharynx [10] S6-688 Nasopharynx [23] Bc5 Nasopharynx [20] RH4 Blood [53] RH6 Blood [53] R14 Unknown [10] R4 Unknown [10] SO-1914 Tympanic cavit^ / as pirate [23' Note: The strains CIO/ R4 did not have the uspAl gene, whereas F16, R14, Z14 lacked the uspA2 gene. [10] The remaining strains contained both uspAl and A2 genes (data not shown). DNA method To detect the presence uspAl, A2 f and A2H genes in those strains which this was unknown, primers and PCR conditions as described by Meier et al. was used. [50] Partial sequencing was also carried out with the UspAl299"452 and UspA2165~31B 5' and 3' primers of the respective uspAl and uspA2 gene of RH4 and BBH18. Confirmation of the presence of the amino acid residues "DQKADIDNNINNIYELAQQQDQHSSDIKTLK" was also performed by PCR with a primer (5'- CAAAGCTGACATCCAAGCACTTG-3') designed from the 5' end of this sequence and 3' primers for uspAl and A2 as described by Meier at al.[50] Recombinant proteins construction and expression Recombinant UspAl50"770 and UspA230"539, which are devoid of their hydrophobic C-termini, has recently been described.[58] The genomic DNA was extracted from M. catarrhalis Bc5 using a DNeasy tissue kit (Qiagen, Hilden, Germany). In addition, recombinant proteins corresponding to multiple regions spanning UspAl50'770 and UspA230'539 were also constructed by the same method- The primers used are listed in table 8. All constructs were sequenced according to standard methods. Expression and purification of the recombinant proteins were done as described previously.[59] Proteins were purified using columns containing a nickel resin (Novagen) according to the manufacturer's instructions for native conditions. The recombinant proteins were analyzed on SDS-PAGE as described.[21] Table 8. Primers used in this present study Antibodies Rabbit anti-UspAl/A2 polyclonal antibodies (pAb) were recently described in detail.[58] The other antibodies used were rabbit anti-human fibronectin pAb, swine FITC-conjugated anti-rabbit pAb, swine horseradish peroxidase (HRP) conjugated anti-rabbit pAb and finally a mouse anti-human CD54 (ICAM1) monoclonal antibody (mAb). Antibodies were from Dakopatts (Glostrup, Denmark). Flow cytometry analysis The UspAl/A2-protein expression and the capacity of M. catarrhalis to bind fibronectin were analyzed by flow cytometry. M. catarrhalis wild type strains and UspAl/A2-deficient mutants were grown overnight and washed twice in phosphate buffered saline containing 3 % fish gelatin (PBS-gelatin) . The bacteria (10B) were then incubated with the anti-UspAl/A2 antiserum or 5 \xg fibronectin (Sigma, St Louis, MO). They were then washed and incubated for 30 min at room temperature (RT) with FITC-conjugated anti-rabbit pAb (diluted according to the manufacturer's instructions) or with 1/100 dilution of rabbit anti-human fibronectin pAb (if fibronectin was first added) for 30 min at RT before incubation with the FITC-conjugated anti-rabbit pAb, After three additional washes, the bacteria were analyzed by flow cytometry (EPICS, XL-MCL, Coulter, Hialeah, FL). All incubations were kept in a final volume of 100 pi PBS-gelatin and the washings were done with the same buffer. Anti-fibronectin pAb and FITC-conjugated anti-rabbit pAb were added separately as a negative control for each strain analyzed. Fibronectin inhibition studies were carried out by pre-incubating 0.25 pmoles of UspA fragments for 1 h with 2 lag of fibronectin before incubation with M. catarrhalis bacteria (10B) . The residual free amount of fibronectin that bound to M. catarrhalis was determined by flow cytometry as outlined above. Binding of M. catarrhalis to immobilized fibronectin Glass slides were coated with 30 pi aliquots of fibronectin (1 mg/ml) and air dried at RT. After washing once with PBS, the slides were incubated in Petri dishes with pre-chilled bacteria at late exponential phase (optical density (OD) at 600 nm = 0.9). After 2 h at RT, glass slides were washed once with PBS followed by Gram staining. Protein labeling and radio immunoassay (RIA) Fibronectin was Iodine labeled (Amersham, Buckinghamshire, England) to a high specific activity (0.05 mol iodine per mol protein) with the Chloramine T method.[21] M. catarrhalis strains BBH18 and RH4 together with their corresponding mutants were grown overnight on solid medium and were washed in PBS with 2 % bovine serum albumin (BSA). Bacteria (108) were incubated for 1 h at 37°C with 125I-labeled fibronectin (1600 kcpm/sample) in PBS containing 2 % BSA. After three washings with PBS 2 % BSA, 125I-labeled fibronectin bound to bacteria was measured in a gamma counter (Wallac, Espoo, Finland). Enzyme-linked immunosorbent assay (ELISA) Microtiter plates (Nunc-Immuno Module; Roskilde, Denmark) were coated with 40 nM of purified recombinant UspAl50"770 and UspA230"539 proteins in 75 mM sodium carbonate, pH 9.6 at 4 °C overnight. Plates were washed four times with washing buffer (50 mM Tris-HCl, 0.15 M NaCl, and 0.1 % Tween 20, pH 7.5) and blocked for 2 h at RT with washing buffer containing 3 % fish gelatin. After four additional washings, the wells were incubated for 1 h at RT with fibronectin (120 pg/ml) diluted in three-fold step in 1.5 % fish gelatin (in wash buffer). Thereafter, the plates were washed and incubated with rabbit anti-human fibronectin pAb for 1 h. After additional washings, HRP-conjugated anti-rabbit pAb was added and incubated for 1 h at RT. Both the antihuman fibronectin and HRP-conjugated anti-rabbit pAb were diluted 1:1,000 in washing buffer containing 1.5 % fish gelatin. The wells were washed four times and the plates were developed and measured at OD450. ELISAs with truncated proteins spanning UspAl50'770 and UspA230's39 were performed with fixed doses of fibronectin at 80 pg/ml and 120 pg/ml, respectively. Cell line adherence inhibition assay Chang conjunctival cells (ATCC CCL 20.2) were cultured in RPMI 1640 medium (Gibco BRL, Life Technologies, Paisley, Scotland) supplemented with 10 % fetal calf serum, 2 mM L-glutamine, and 12 pg of gentamicin/ ml. On the day before adherence inhibition experiments, cells were harvested, washed twice in gentamicin-free RPMI 1640, and added to 96 well tissue culture plates (Nunc) at a final concentration of 104 cells/ well in 200 ]xl of gentamicin-free culture medium. Thereafter, cells were incubated overnight at 37 °C in a humidified atmosphere of 5 % CO2and 95 % air. On the day of experiments, inhibition of M. catarrhalis adhesion was carried out by pre-incubating increasing concentration of recombinant UspAl/A2 truncated proteins containing the fibronectin binding domains (UspAl299"432 and UspA2165"3ie) or rabbit anti-human fibronectin pAb (diluted 1:50) for 1 h. Nonfibronectin binding recombinant proteins (UspAl433*"580 and UspA230"177) were used as controls, Chang epithelial cells are known to express ICAM1.[18] Hence an anti-ICAMl antibody was used to differentiate if the inhibitory effect of the anti-fibronectin antibody was secondary to steric hindrance. Subsequently, M. catarrhalis RH4 (106) in PBS-gelatin was inoculated onto the confluent monolayers. In all experiments, tissue culture plates were centrifuged at 3,000 x g for 5 min and incubated at 37 °C in 5 % C02. After 30 min, infected 'monolayers were rinsed several times with PBS-gelatin to remove non-adherent bacteria and were then treated with trypsin-EDTA (0.05 % trypsin and 0.5 mM EDTA) to release the Chang cells from the plastic support. Thereafter, the resulting cell/ bacterium suspension was seeded in dilution onto agar plates containing BHI and incubated overnight at 37 °C in 5 % CO2. Determination of fibronectin expression in Chang conjunctival epithelial cells Chang conjunctival epithelial cells were harvested by scraping followed by re-suspension in PBS-gelatin. Cells (1 x 106 /ml) were labeled with rabbit anti-human fibronectin pAb followed by washing and incubation with a FITC- conjugated anti-rabbit pAb. After three additional washes, the cells were analyzed by flow cytometry as outlined above. Interaction between M. catarrhalis and laminin Bacterial strains and culture conditions The clinical M* catarrhalis strains BBH18 and RH4 and their corresponding mutants were previously described.[58] Both strains have a relatively higher expression of UspA2 compared to UspAl.[58] The mutants expressed equal amount of M. catarrhalis immunoglobulin D-binding protein (MID) when compared to wild type strains. Bacteria were routinely cultured in brain heart infusion (BHI) broth or on BHI agar plates at 37 °C. The UspAl-deficient, UspA2-deficient and double mutants were cultured in BHI supplemented with antibiotics as described.[58] Recombinant protein construction and expression Recombinant UspAl50"770 and UspA230"539, which are devoid of their hydrophobic C-termini, were manufactured.[58] In addition, recombinant proteins corresponding to multiple regions, spanning UspAl50"770 and UspA230"539 were used. [78] Antibodies Rabbit anti-UspAl/A2 and anti-MID polyclonal antibodies (pAb) were used.[22, 58] Rabbit anti-laminin pAb was from Sigma (St Louis, MO, USA). Swine horseradish peroxidase (HRP)-conjugated anti-rabbit pAb was from Dakopatts (Glostrup, Denmark). Binding of M. catarrhalis to immobilized laminin Microtiter plates (Nunc-Immuno Module; Roskilde, Denmark) were coated with Engelbreth-Holm-Swarm mouse sarcoma laminin (Sigma, Saint Louis, USA) or bovine serum albumin (BSA) (30 yig/ml) in Tris-HCL, pH 9.0 at 4°C overnight. The plates were washed with phosphate buffered saline and 0.05% Tween 20, pH 7.2 (PBS-Tween) and subsequently blocked with 2 % BSA in PBS + 0.1 % Tween 20, pH 7.2. M. catarrhalis RH4 and BBH18 (108) in 100 jil were then added followed by incubation for 1 h. Unbound bacteria were removed by washing 3 times with PBS-Tween. Residual bound bacteria were detected by means of an anti-MID pAb, followed by detection with HRP-conjugated anti-rabbit pAb. The plates were developed and measured at OD450 according to a standard protoccol. Enzyme-linked immunosorbent assay (ELISA) Microtiter plates (Nunc-Immuno Module) were coated with 40 nM of purified recombinant UspAl50~770 and UspA230"539 proteins in 75 mM sodium carbonate, pH 9.6 at 4°C. Plates were washed four times with washing buffer (50 mM Tris-HCl, 0.15 M NaCI, and 0.1 % Tween 20, pH 7.5) and blocked at RT with washing buffer containing 3 % fish gelatin. After additional washings, the wells were incubated for 1 h at RT with laminin at different dilutions as indicated in 1.5 % fish gelatin (in wash buffer). Thereafter, the plates were washed and incubated with rabbit anti-laminin pAb. After additional washings, HRP-conjugated anti-rabbit pAb was added and incubated at RT. Both the anti-laminin and HRP-conjugated anti-rabbit pAb were diluted 1:1,000 in washing buffer containing 1.5 % fish gelatin. The wells were washed and the plates were developed and measured at OD450. Uncoated wells incubated with identical dilutions of laminin were used as background controls. ELISAs with truncated proteins spanning UspAl50"770 and UspA230"539 were performed with fixed doses of laminin (20 yg/ml) . Interaction between AT. catarrhalis and C3 and C3met Bacterial strains and culture conditions The clinical M. catarrhalis isolates and related subspecies have recently been described in detail.[21, 53] Type strains were from the Culture Collection, University of Gothenburg (CCUG; Department of Clinical Bacteriology, Sahlgrenska Hospital, Gothenburg, Sweden), or the American Type Culture Collection (ATCC; Manassas, Va); Neisseria gonorrheae CCUG 15821, Streptococcus pyogenes CCUG 25570 and 25571, Streptococcus agalactiae CCUG 4208, Streptococcus pneumoniae ATCC 49619, Legionella pneumophila ATCC 33152, Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 29213, and finally Staphylococcus aureus ATCC 25923. The remaining strains in Table 9 were clinical isolates from Medical Microbiology, Department of Laboratory Medicine, Malmo University Hospital, Lund University, Sweden. The different non-moraxella species were grown on appropriate standard culture media, M. catarrhalis strains were routinely cultured in brain heart infusion (BHI) liquid broth or on BHI agar plates at 37°C. M. catarrhalis BBH18 and RH4 mutants were manufactured as previously described. [22, 23, 58] The MID-deficient mutants were grown in BHI containing 50 |ag/ml kanamycin. The UspAl-deficient mutants were cultured in BHI supplemented with 1.5 |ig/ml chloramphenicol (Sigma, St. Louis, MO), and UspA2-deficient mutants were incubated with 7 |xg/ml zeocin (Invitrogen, Carlsbad, CA) . Both chloramphenicol and zeocin were used for growth of the UspAl/ A2 double mutants. Antibodies Rabbits were immunized intramuscularly with 200 |ig recombinant full-length UspAl emulsified in complete Freunds adjuvant (Difco, Becton Dickinson, Heidelberg, Germany) , and boosted on days 18 and 36 with the same dose of protein in incomplete Freunds adjuvant.[22] Blood was drawn 3 weeks later. To increase the specificity, the anti-UspAl antiserum was affinity-purified with Sepharose-conjugated recombinant UspAl50"170. [58] The antiserum bound equally to UspAl and UspA2 and was thus designated anti-UspAl/ A2 pAb. The rabbit anti-human C3d pAb and the FITC-conjugated swine anti-rabbit pAb were purchased from Dakopatts (Glostrup, Denmark), and the goat anti-human C3 were from Advanced- Research Technologies (San Diego, CA). The horseradish peroxidase (HRP)-conjugated donkey anti-goat pAb was obtained from Serotec (Oxford, UK). Proteins and iodine labelling The manufacture of recombinant UspAl50"770 and UspA230*539, which are devoid of their hydrophobic C-termini, has recently been described.[23] The truncated UspAl and UspA2 proteins were manufactured as described in detail by Tan et al. [78] C3b was purchased from Advanced Research Technologies. C3(H20) was obtained by freezing and thawing of purified C3. The C3b-like molecule (C3met) was made by incubation of purified C3 with 100 mM methylamine (pH 8.0) for 2 h at 37°C, and subsequent dialysis against 100 mM Tris-HCl (pH 7.5), 150 mM NaCl. For binding studies, C3met was labelled with 0.05 mol 125I (Amersham, Buckinghamshire, England) per mol protein, using the Chloramine T method-[25] Flow cytometry analysis Binding of C3 to M. catarrhalis and other species was analyzed by flow cytometry. Bacteria were grown on solid medium overnight and washed twice in PBS containing 2 % BSA (Sigma) (PBS-BSA). Thereafter, bacteria (108 colony forming units; cfu) were incubated with C3met, C3b, C3 (H20), or 10 % NHS with or without 10 mM EDTA or 4 mM MgCl2 and 10 mM EGTA (Mg-EGTA) in PBS-BSA for 30 min at 37°C. After washings, the bacteria were incubated with anti-human C3d pAb for 30 min on ice, followed by washings and incubation for another 30 min on ice with FITC-conjugated goat anti-rabbit pAb. After three additional washes, bacteria were analyzed by flow cytometry (EPICS, XL-MCL, Coulter, Hialeah, FL). All incubations were kept in a final volume of 100 jxl PBS-BSA and the washings were done with the same buffer. The anti-human C3d pAb and FITC-conjugated anti-rabbit pAb were added separately as a negative control for each strain analyzed. In the inhibition studies, serum was preincubated with 100 nM of the recombinant UspAl50"770 and UspA230"539 proteins for 30 min at 37°C. To analyze the characteristics of the M.. catarrhalxs and C3 interaction, increasing concentrations of NaCl (0 - 1.0 M) was added to bacteria and C3met. To analyze UspAl/ A2 expression, bacteria (108 cfu) were incubated with the anti-UspAl/ A2 pAb and washed as described above. A FITC-conjugated goat anti-rabbit pAb diluted according to the manufacturers instructions was used for detection. To assure that EDTA did not disrupt the outer membrane proteins UspAl and UspA2, M. catarrhalis was incubated with or without EDTA followed by detection of UspAl/ A2 expression. EDTA,' at the concentrations used in the NHS-EDTA experiments, did not change the density of UspAl/A2. Serum and serum bactericidal assay Normal human serum (NHS) was obtained from five healthy volunteers- The blood was allowed to clot for 30 min at room temperature and thereafter incubated on ice for 60 min. After centrifugation, sera were pooled, aliquoted and stored at -70°C. To inactivate both the classical and alternative pathways, 10 mM EDTA was added. In contrast, Mg-EGTA was included to inactivate the classical pathway. Human serum deficient in the C4BP was prepared by passing fresh serum through a HiTrap column (Amersham Biosciences) coupled with mAb 104, a mouse mAb directed against CCP1 of the cc-chain of C4BP.[41] The flow through was collected and the depleted serum was stored in aliquots at -70°C. Serum depleted of Clq was obtained via the first step of Clq purification [79] using Biorex 70 ion exchange chromatography (Bio-Rad, Hercules, CA). The resulting sera displayed normal haemolytic activity. The factor D and properdin deficient serum was kindly provided by Dr. Anders SjSholm (Department of Medical Microbiology, Lund University, Lund, Sweden). M. catarrhalis strains were diluted in 2,5 mM Veronal buffer, pH 7.3 containing 0.1 % (wt/vol) gelatin, 1 mM MgCl2, 0.15 mM CaCl2, and 2.5 % dextrose (DGVB4**) . Bacteria (103 cfu) were incubated together with 10 % NHS and EDTA or Mg-EGTA in a final volume of 100 jxl. The bacteria/ NHS was incubated at 37°C and at various time points, 10 fxl aliquots were removed and spread onto BHI agar plates. In inhibition studies, 10 % serum was incubated with 100 nM of the recombinant UspAl50"770 and UspA230"539 proteins for 30 min at 37°C before bacteria were added. Dot blot assays Purified recombinant UspAl50'770 and UspA230"539 diluted in three-fold steps (1.9 - 150 nM) in 100 \xl of 0,1 M Tris-HCl, pH 9.0 were applied to nitrocellulose membranes (Schleicher & Schull, Dassel, Germany) using a dot blot device. After saturation, the membranes were incubated for 2 h with PBS-Tween containing 5 % milk powder at room temperature and washed four times with PBS-Tween. Thereafter, 5 kcpm [125I]-labelled C3met in PBS-Tween with 2 % milk powder was added overnight at 4°C. The bound protein was visualized with a Personal FX (Bio-Rad) using intensifying screens. Surface plasmon resonance (Biacore) The interaction between UspAl50"770 or UspA230*539 and C3 was further analysed using surface plasmon resonance (Biacore 2000; Biacore, Uppsala, Sweden) as recently described for the UspAl/2-C4BP interaction.[58] The KD (the equilibrium dissociation constant) was calculated from a binding curve showing response at equilibrium plotted against the concentration using steady state affinity model supplied by Biaevaluation software (Biacore). Enzyme-linked immunosorbent assay (ELISA) Microtiter plates (Nunc-Immuno Module; Roskilde, Denmark) were coated with triplets of purified recombinant UspAl50"770, UspA230"539, or the truncated UspAl and UspA2 fragments (40 nM in 75 mM sodium carbonate, pH 9.6) at 4°C overnight. Plates were washed four times with washing buffer (PBS with 0.1 % Tween 20, pH 7.2) and blocked for 2 hrs at room temperature with washing buffer supplied with 1.5 % ovalbumin (blocking buffer). After washings, the wells were incubated overnight at 4°C with 0.25 fig C3met in blocking buffer. Thereafter, the plates were washed and incubated with goat anti-human C3 in blocking buffer for 1 h at RT. After additional washings, HRP-conjugated donkey anti-goat pAbs was added for another 1 h at RT. The wells were washed four times and the plates were developed and measured at OD450. Haernolytic assay Rabbit erythrocytes were washed three times with ice-cold 2,5 mM Veronal buffer, pH 7.3 containing 0.1 % (wt/vol) gelatin, 7 mM MgCl2, 10 mM EGTA, and 2.5 % dextrose (Mg++EGTA) , and resuspended at a concentration of 0.5 x 109 cells/ml. Erythrocytes were incubated with various concentrations (0 to 4 %) of serum diluted in Mg++EGTA. After 1 h at 37°C, erythrocytes were centrifuged and the amount of lysed erythrocytes was determined by spectrophotometry measurement of released hemoglobin at 405 nm. For inhibition with UspAl and UspA2, 10 % serum was. preincubated with 100 nM of recombinant UspAl50-770 and/ or UspA230"539 proteins for 30 min at 37°C, and thereafter added to the erythrocytes at 0 'to 4 %. -' ' Isolation of polymorphonuclear leukocytes and phagocytosis Human polymorphonuclear leukocytes (PMN) were isolated from fresh blood of healthy volunteers using macrodex (Pharmaiink AB, Upplands Vasby, Sweden). The PMN were centrifuged for 10 min at 300g, washed in PBS and resuspended in RPMI 1640 medium (Life Technologies, Paisley, Scotland). The bacterial suspension (0.5 x 108) was opsonized with 3 % of either NHS or NHS-EDTA, or 20 |xg of purified C3met for 15 min at 37DC, After washes, bacteria were mixed with PMN (1 x 107 cells/ml) at a bacteria/PMN ratio of 10:1 followed by incubation at 37°C with end-over-end rotation. Surviving bacteria after 0, 30, 60, and 120 min of incubation was determined by viable counts. The number of engulfed NHS-treated bacteria was compared with bacteria phagocytosed in the absence of NHS. S. aureus opsonized with NHS was used as positive control. Examples and results Interaction between M. catarrhalis and fibronectin M. catarrhalis devoid of UspAl and A2 does not bind soluble or immobilized fibronectin. We selected a random series of M. catarrhalis clinical strains (n=13) (table 7) and tested them for fibronectin binding in relation to their UspAl/A2 expression by flow cytometry analysis. High UspAl/A2 expression as determined by high mean fluorescence intensity (MFI) was correlated to UspAl/A2 expression (Pearson correlation coefficient 0.77, P Two M. catarrhalis isolates (BBH18 and RH4) and their specific mutants lacking UspAl, UspA2 or both proteins were also analyzed by flow cytometry. M. catarrhalis BBH18 strongly bound fibronectin with a mean fluorescence intensity (MFI) of 96.1 (figure IF). In contrast, BBRlQAuspAl showed a decreased fibronectin binding with an MFI of 68.6 (figure 1G) . Fibronectin binding to BBH18AuspA2 and the double mutant BBR1BAuspAl/A2 revealed an MFI of only 10.7 and 11.5, respectively (figure \K, II). Similar results were obtained with UspAl/A2 mutants of the clinical strain M. catarrhalis RH4. Taken together, these results suggest that UspAl and A2 bound fibronectin and that the ability of the bacteria to bind fibronectin strongly depended on UspAl/A2 expression. To further analyze the interaction between fibronectin and M. catarrhalisf 125I-labeled fibronectin was incubated with two clinical M. catarrhalis isolates (BBH18 and RH4) and their respective mutants. The wild type M. catarrhalis RH4 strongly bound 125I-fibronectin while the corresponding AuspAl mutant showed 80 % binding of the wild type. In contrast, the AuspA2 and double mutant bound a25I-fibronectin at 14 % and 12 %, respectively, which was just above the background levels (5,0 to 10 %] (figure 2). Similar results were obtained with M. catarrhalis BBH18 and the corresponding UspAl/A2 mutants. Thus, our results suggest that both UspAl and A2 are required for the maximal binding of soluble fibronectin by M. catarrhalis. To investigate the bacterial attachment to immobilized fibronectin, M. catarrhalis RH4 and its corresponding AuspAl/A2 mutants were applied onto fibronectin coated glass slides. After 2 h of incubation, slides were washed, and subsequently Gram stained. M. catarrhalis wild type and the AuspAl mutant were found to strongly adhere to the fibronectin coated glass slides (figure 3A and 3B) . In contrast, M. catarrhalis AuspA2 and AuspAl/A2 double mutants weakly adhered to the fibronectin coated glass slide with only a few bacteria left after washing (figure 3C and 3D, respectively). Experiments with another M. catarrhalis clinical isolate (BBH18) and its derived mutants showed a similar pattern indicating that UspA2 was of major importance for AT. catarrhalis binding to immobilized fibronectin. The fibronectin binding domains include amino acid residues located between 299 and 452 of UspAl and between 165 and 318 of UspA2 To further analyze the interactions of UspAl and A2 with fibronectin, truncated UspAl50'770 and UspA230"539 were recombinantly produced in E. coli, coated on microtiter plates and incubated with increasing concentrations of fibronectin. Bound fibronectin was detected with an anti-human fibronectin pAb followed by incubation with a horseradish peroxidase conjugated anti-rabbit pAb. Both recombinant UspAl50"770 and UspA230"539 bound soluble fibronectin and the interactions were dose-dependent (figure 4) , To define the fibronectin-binding domain of UspAl, recombinant proteins spanning the entire molecule of UspAl50" 770 were manufactured. Fibronectin was incubated with the immobilized UspAl proteins fragments and the interactions were quantified by ELISA. UspAl50"491 bound fibronectin almost as efficiently as UspAl50"770 suggesting that the binding domain was within this part of the protein. Among the other truncated fragments, UspAl299"452 efficiently bound fibronectin (figure 5 A) . In parallel, the interactions between fibronectin and several recombinant UspA2 fragments including amino acids UspA230"539 were analyzed. The two fragments UspA2m~31B and UspA2165"318 strongly bound fibronectin (figure 5B). Our findings provide significant evidence that the binding domains include residues found within UspAl299"452 and UspA2165"318. A sequence comparison between these two binding fragments revealed that the 31 amino acid residues "DQKADIDNNINNIYELAQQQDQHSSDIKTLK" were identical for UspAl and A2 (figure 6), Moreover, this repeat sequence was also found in the uspAl and A2 gene of M. catarrhalis BBH18 and RH4 (data not shown). UspAl50"491 and UspAl299"452 fragments competitively inhibit M. catarrhalis fibronectin binding To further validate our findings on the UspAl/A2 fibronectin binding domains, recombinant truncated UspAl proteins were tested for their capacity to block fibronectin binding to AT. catarrhalis. Fibronectin (2 pg) was pre-incubated with 0.25 pmoles of recombinant UspAl fragments and subsequently incubated with M. catarrhalis. Finally, M. catarrhalis UspA-dependent fibronectin binding was measured by flow cytometry. Pre-incubation with UspAl50"491 and UspAl299"452 resulted in decreased fibronectin binding with a 95 % reduction for UspAl50*491 and a 63 % reduction for UspAl299"452 (figure 7), When fibronectin was pre-incubated with the truncated UspA2101"318 , an inhibition of 50% was obtained. Thus, the fibronectin binding domains of UspAl and A2 block the interactions between fibronectin and M. catarrhalis. UspAl299"452 and UspA2165"318 inhibit M. catarrhalis adherence to Chang epithelial cells Epithelial cells are known to express fibronectin and many bacteria attach to epithelial cells via cell-associated fibronectin.[46, 54, 69, 77] Previous studies have shown that M. catarrhalis adhere to epithelial cells.[43, 49] We analyzed Chan conjunctival cells, which have frequently been used in adhesion experiments with respiratory pathogens. Chang cells strongly expressed fibronectin as revealed by flow cytometry analysis (figure 8A). To analyze whether the UspA-dependent fibronectin binding was important for bacterial adhesion, Chang epithelial cells were pre-incubated with anti-human fibronectin pAb, or the recombinant proteins UspAl299"452 and UspA2165"318. Thereafter, M. catarrhalis RH4 was added and bacterial adhesion analyzed. The relative adherence (measured by the number of colony forming units) after preincubation with 0.4 pinoles per 200 pi of UspAl299"452, UspA2165~318, or an anti-human fibronectin pAb were 36 %, 35 % and 32%, respectively. Higher concentrations of recombinant peptides did not result in further inhibition. In contrast, the non-fibronectin binding fragments UspAl433"580 and UspA230~ 177 did not inhibit the interactions between M. catarrhalis and the Chang epithelial cells (figure BE). Thus, fibronectin on Chang epithelial cells may function as a receptor for M. catarrhalis and the amino acid residues 299-452 of UspAl and 165-318 of UspA2 contain the ligand responsible for the interactions. Interaction between M. catarrhalis and laminin M. catarrhalis binds laminin through UspAl and A2 Two clinical M. catarrhalis isolates (BBH18 and RH4) and their specific mutants lacking UspAl, UspA2 or both proteins were analyzed by a whole-cell ELISA. M. catarrhalis RH4 strongly bound to immobilized laminin. (figure 9A) . In contrast, M. catarrhalis RH4 uspAl mutant (RH4£uspAl) showed a laminin binding of 89.9% of the wild type. M. catarrhalis RH4 uspA2 mutant {RK4AuspA2) and the double mutant RH4AuspAl/A2 15.2% and 18.1% binding capacity of the wild type, respectively. This was not significantly different from the residual adhesion to BSA coated plates. Similar results were obtained with UspAl/A2 mutants originating from the clinical strain M. catarrhalis BBH18. In these two strains (BBH18 and RH4), UspA2 is the predominant protein expressed as compared to UspAl, explaining the minimal difference in binding between the wild type and RRAAuspAl. Taken together, these results show that UspAl and A2 bound laminin. To further analyze the binding between UspAl/A2 and laminin, truncated UspAl50"*770 and UspA23D"539 were produced in E. coli. Recombinant proteins were coated on microtiter plates and incubated with increasing concentrations of laminin. Bound laminin was detected with a rabbit anti-laminin pAb followed by incubation with an HRP-conjugated anti-rabbit pAb. Both recombinant UspAl50"770 and UspA230"539 strongly bound soluble laminin and the binding was dose-dependent and saturable (figure 9B). To define the laminin binding domains, recombinant UspAl and A2 spanning the entire molecules were manufactured. Laminin was incubated with immobilized truncated UspAl and A2 fragments and followed by quantification by ELISA, UspAl50"491 bound to laminin almost as efficiently as UspAl50"770 suggesting that the binding domain was within this part of the protein. However, among the other truncated fragments spanning this region, no other fragment appeared to bind laminin. The N-terminal part, UspA230"351, was able to retain 44.7 % binding capacity as compared to the full length protein. The shorter protein UspA230"177 showed a 43.7 % binding capacity, (figure 10B) . These results show that the binding domains include residues found within the N-terminals of both UspAl and UspA2. Interaction between M. catarrhalis and C3 and C3met M. catarrhalis outer membrane proteins UspAl and UspA2 inhibit both the classical and the alternative pathway of the complement cascade conclusion, M, catarrhalis has developed sophisticated ways of combating both the humoral and innate immune systems. 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Evasion of pathogens by avoiding recognition or eradication by complement, in part via molecular mimicry, Mol. Immunol. 36:249-260. Zipfel, P. F., C. Skerka, J. Hellwage, S. T. Jokiranta, S. Meri, V, Brade, P. Kraiczy, M. Noris, and G. Remuzzi. 2001. Factor H family proteins: on complement, microbes and human diseases, Biochem. Soc. Trans. 30:971-978. CLAIMS 1. Peptide consisting of Sequence ID No. 1, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 2. Peptide consisting of Sequence ID No. 2, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 3. Peptide consisting of Sequence ID No. 3, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 4. Peptide consisting of Sequence ID. No. 4, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 5. Peptide consisting of Sequence ID No. 5, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. '6. Peptide consisting of Sequence ID No. 6, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 7. Peptide consisting of Sequence ID No. 7, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 8. Peptide consisting of Sequence ID No. 8, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 9. .Peptide consisting of Sequence ID No. 9, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 10. Peptide consisting of Sequence ID No. 10, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 11. Use of at least one peptide according to any one of claims 1 to 10 for the production of a medicament for the treatment or prophylaxis of an infection. 12. Use according to claim 11, wherein the infection is caused by Moraxella catarrhalis. 13. Use according to claim 11 or 12, for the prophylaxis or treatment of otitis media, sinusitis or lower respiratory tract infections. 14. A ligand comprising a fibronectin binding domain, said ligand consisting of an amino acid sequence selected from the group consisting of Sequence ID No. 1 to Sequence ID No. 3, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 15. A ligand comprising a laminin binding domain, said ligand consisting of an amino acid sequence selected from the group consisting of Sequence ID No. 4 to Sequence ID No. 8, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 16. A ligand comprising a C3 or C3met binding domain, said ligand consisting of an amino acid sequence selected from the group consisting of Sequence ID No, 4, Sequence ID No. 6, Sequence ID No. 9 and Sequence ID No. 10, or a fragment, homologue, functional equivalent, derivative, degenerate or hydroxylation, sulphonation or glycosylation product or other secondary processing product thereof. 17. A medicament comprising one or more ligands according to any one of claims 14 to 16 and one or more pharmaceutically acceptable adjuvants, vehicles, excipients, binders, carriers, or preservatives. 18. A vaccine comprising one or more ligands according to any one of claims 14 to 16 and one or more pharmaceutically acceptable adjuvants, vehicles, excipients, binders, carriers, or preservatives. 19. A method of treating or preventing an infection in an individual comprising administering a pharmaceutically effective amount of a medicament or a vaccine according to claim 17 or 18. 20. A method according to claim 19, wherein the infection is caused by Moraxella catarrhalis. 21. A nucleic acid sequence encoding a ligand, protein or peptide of the present invention, as well as homologues, polymorphisms, degenerates and splice variants thereof. 22. A polypeptide or a polypeptide truncate comprising at least one of the conserved sequences of Sequence ID No 1 to Sequence ID No 3 with the ability of binding fibronectin. 23. A polypeptide or a polypeptide truncate comprising at least one of the conserved sequences of Sequence ID No 4 to Sequence ID No 8 with the ability of binding laminin. 24. A polypeptide or a polypeptide truncate comprising at least one of the conserved sequences of Sequence ID No 4, 6, 9, and 10, with the ability of binding C3 and/or C3met. 25. A vaccine composition comprising an effective amount of UspAl and/or UspA2 in combination with an effective amount of protein MID and one or more pharmaceutically acceptable adjuvants, vehicles, excipients, binders, carriers, or preservatives. 26. Use of an effective amount of UspAl and/or UspA2 in combination with an effective amount of protein MID for the production of a medicament for the treatment or prophylaxis of an infection. 27. Use according to claim 26, wherein the infection is caused by Moraxella catarrhalis. |
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| Patent Number | 272402 | ||||||||||||
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| Indian Patent Application Number | 724/CHENP/2008 | ||||||||||||
| PG Journal Number | 14/2016 | ||||||||||||
| Publication Date | 01-Apr-2016 | ||||||||||||
| Grant Date | 31-Mar-2016 | ||||||||||||
| Date of Filing | 11-Feb-2008 | ||||||||||||
| Name of Patentee | ARNE FORSGREN AB | ||||||||||||
| Applicant Address | SOTHONSVAGEN 4 B, S-230 11 FALSTERBO, SWEDEN. | ||||||||||||
Inventors:
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| PCT International Classification Number | CO7K14/21 | ||||||||||||
| PCT International Application Number | PCT/SE2006/000931 | ||||||||||||
| PCT International Filing date | 2006-08-08 | ||||||||||||
PCT Conventions:
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