Patent Publication Number: US-10786562-B2

Title: Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 15/486,513, filed Apr. 13, 2017, which is a divisional of U.S. patent application Ser. No. 14/986,104, filed Dec. 31, 2015 and now issued as U.S. Pat. No. 9,657,067, which is a divisional of U.S. patent application Ser. No. 14/508,033, filed Oct. 7, 2014, and now issued as U.S. Pat. No. 9,255,127, which is a divisional of U.S. patent application Ser. No. 13/666,941, filed Nov. 1, 2012, and now issued as U.S. Pat. No. 8,895,030, which is a divisional of U.S. patent application Ser. No. 13/314,727, filed Dec. 8, 2011, and now issued as U.S. Pat. No. 8,323,667, which is a divisional of U.S. application Ser. No. 12/063,408, filed on Feb. 8, 2008, and now issued as U.S. Pat. No. 8,092,811, which is a national stage filing under 35 U.S.C. § 371 of International Application No. PCT/SE2006/000931, filed on Aug. 8, 2006, which claims the benefit of priority of U.S. Provisional Application No. 60/706,745, filed on Aug. 10, 2005, and of U.S. Provisional Application No. 60/707,148, filed on Aug. 11, 2005. All prior applications are incorporated herein by reference in their entireties. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing, which is hereby incorporated by reference in its entirety. A computer readable copy of the Sequence Listing (ASCII copy) is submitted concurrently herewith to the U.S. Patent and Trademark Office via EFS-Web as part of a file created on created on Nov. 1, 2012, named SequenceListing.txt, and being 199,367 bytes in size. 
     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  Haemphilus 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 UspA1 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 UspA1/A2 proteins together with smaller fragments spanning the entire molecule have been tested according to the present invention for fibronectin binding. Both UspA1 and A2 bound fibronectin and the fibronectin binding domains were found to be located within UspA1 299-452  and UspA2 165-318 . 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  UspA1 and A2 are involved in the adherence to epithelial cells via cell-associated fibronectin. The biologically active sites within UspA1 299-452  and UspA2 165-318  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) A1 and A2 also bind to laminin. Laminin binding domains of UspA1 and A2 were, amongst others, found within the N-terminal halves of UspA1 50-491  and UspA2 30-351 . These domains are also containing the fibronectin binding domains. However, the smallest fragments that bound fibronectin, UspA1 299-452  and UspA2 165-318 , did not bind laminin to any appreciable extent. Fragments smaller than the N-terminal half of UspA1 (UspA1 50-491 ) lose all its laminin binding ability, whereas with UspA2, only UspA2 30-170  bound laminin albeit at a lower level than the whole recombinant protein (UspA2 30-539 ). These findings suggest that different parts of the molecule might have different functional roles. UspA1 50-770  was also found to have laminin binding properties. 
     Comparing the smallest laminin binding regions of UspA1 and A2, we find that there is, however, little similarity by way of amino acid homology between UspA2 30-170  and UspA1 50-491  (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 UspA1 50-770  and UspA2 30-539  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 A1 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 mg/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) (Cys 1010 ) and a glutamyl carbonyl (Gln 1012 ) on the C3 α-chain. [76] Proteolytic cleavage of a 77-residue peptide from the amino terminus of the C3 α-chain generates C3a (anaphylatoxin) and C3b. Attachment of C3b is then accomplished through a covalent link between the carbonyl group of the metastable thioester and either —NH 2  or —OH groups of proteins or carbohydrate structures on the activator surface. [36, 37]  M. catarrhalis  UspA1 and A2 have been found to non-covalently and in a dose dependent manner bind both the third component of complement (C3) from EDTA-treated serum and methylamine treated C3 (C3met). UspA1 50-770  and UspA2 30-539  have been found to bind to C3 and C3met. The C3-binding region for UspA2 was found to mainly be localised in UspA2 200-458 . UspA1 has however been found to have a minor role in the interactions. The biologically active sites within UspA1 50-770  and UspA2 30-539  are suggested as potential candidates to be included in a vaccine against  M. catarrhalis.    
     The UspA family consists of UspA1 (molecular weight 88 kDa), UspA2 (62 kDa), and the hybrid protein UspA2H (92 kDa).[2, 43] These proteins migrate as high molecular mass complexes in SDS-PAGE, are relatively conserved and hence important vaccine candidates. The amino acid sequences of UspA1 and A2 are 43% identical and have 140 amino acid residues that are 93% identical. [2] In a series of 108  M. catarrhalis  nasopharyngeal isolates from young children with otitis media, uspA1 and uspA2 genes were detected in 107 (99%) and 108 (100%) of the isolates, respectively. Twenty-one percent were identified as having the hybrid variant gene uspA2H.[50] Moreover, it is known that naturally acquired antibodies to UspA1 and A2 are bactericidal.[15] 
     Several functions have been attributed to the UspA family of proteins. UspA1 expression is essential for the attachment of  M. catarrhalis  to Chang conjunctival epithelial cells and Hep-2 laryngeal epithelial cells.[43, 49] In a more recent study, UspA1 was shown to bind carcinoembryonic antigen related cell adhesion molecules (CEACAM) expressed in the lung epithelial cell line A549.[31] Purified UspA1 has also been shown to bind fibronectin in dot blot experiments while purified UspA2 did not.[49] Both UspA1 and A2 may play important roles for  M. catarrhalis  serum resistance.[1, 5, 58, 60] 
     The present invention demonstrates that both UspA1 and A2 are determinants for  M. catarrhalis  binding to fibronectin and laminin in the clinical isolates  M. catarrhalis  BBH18 and RH4. Interestingly, recombinant UspA1 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 UspA1 and 165 to 318 of UspA2. These two domains share 31 amino acid residues sequence identity. Importantly, truncated protein fragments containing these residues in UspA1 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 UspA1/A2 both 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  M. 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  M. catarrhalis , 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 UspA1 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. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Multiple alignment of full length UspA1 protein 
               
               
                 sequences, associated identity percentages 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 O12E 
                 O35E 
                 O46E 
                 P44 
                 TTA24 
                 TTA37 
                 V1171 
               
               
                   
               
               
                 ATCC25238 
                 81 
                 75 
                 83 
                 83 
                 84 
                 79 
                 84 
               
               
                 O12E 
                   
                 74 
                 77 
                 83 
                 76 
                 72 
                 75 
               
               
                 O35E 
                   
                   
                 72 
                 74 
                 83 
                 73 
                 78 
               
               
                 O46E 
                   
                   
                   
                 81 
                 81 
                 82 
                 80 
               
               
                 P44 
                   
                   
                   
                   
                 81 
                 75 
                 77 
               
               
                 TTA24 
                   
                   
                   
                   
                   
                 76 
                 84 
               
               
                 TTA37 
                   
                   
                   
                   
                   
                   
                 78 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 UspA2 Pileup Analysis - Strains and sequences used 
               
            
           
           
               
               
               
               
               
            
               
                   
                 acc 
                 Strain 
                 des 
                 s1 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                     TREMBL: O54407_MORCA 
                 O54407 
                 O35E 
                 Ubiquitous 
                 576 
               
               
                   
                   
                   
                 surface 
                   
               
               
                   
                   
                   
                 protein A 2. 
                   
               
               
                     TREMBL: Q58XP4_MORCA 
                 Q58XP4 
                 MC317 
                 UspA2. 
                 650 
               
               
                     TREMBL: Q848S1_MORCA 
                 Q848S1 
                 E22 
                 Ubiquitous 
                 877 
               
               
                   
                   
                   
                 surface 
                   
               
               
                   
                   
                   
                 protein A2H. 
                   
               
               
                     TREMBL: Q848S2_MORCA 
                 Q848S2 
                 V1122 
                 Ubiquitous 
                 616 
               
               
                   
                   
                   
                 surface 
                   
               
               
                   
                   
                   
                 protein A2. 
                   
               
               
                     TREMBL: Q8GH86_MORCA 
                 Q8GH86 
                 P44 
                 UspA2. 
                 668 
               
               
                     TREMBL: Q9L961_MORCA 
                 Q9L961 
                 TTA37 
                 USPA2H. 
                 889 
               
               
                     TREMBL: Q9L962_MORCA 
                 Q9L962 
                 O46E 
                 USPA2H. 
                 894 
               
               
                     TREMBL: Q9L963_MORCA 
                 Q9L963 
                 O12E 
                 USPA2 
                 684 
               
               
                   
                   
                   
                 (Ubiquitous 
                   
               
               
                   
                   
                   
                 surface 
                   
               
               
                   
                   
                   
                 protein A2). 
                   
               
               
                     TREMBL: Q9XD51_MORCA 
                 Q9XD51 
                 V1171 
                 UspA2. 
                 674 
               
               
                     TREMBL: Q9XD53_MORCA 
                 Q9XD53 
                 TTA24 
                 UspA2. 
                 613 
               
               
                 TREMBL: Q8RTB2_MORCA 
                 Q8RTB2 
                 SP12-5 
                 UspA2 
                 686 
               
               
                     TREMBL: Q9XD55_MORCA 
                 Q9XD55 
                 ATCC25238 
                 UspA2. 
                 630 
               
               
                 Forsgren_UspA2 
                   
                   
                 UspA2. 
                 630 
               
               
                   
               
            
           
         
       
     
     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 UspA1 &amp; 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 UspA1 from  Moraxella   catarrhalis   
               
               
                   
                 strain BC5 (SEQ ID NO: 32): 
               
               
                   
                 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 (SEQ ID NO: 33): 
               
               
                   
                 MKTMKLLPLK IAVTSAMIIG LGAASTANAQ AKNDITLEDL 
               
               
                   
                   
               
               
                   
                 PYLIKKIDQN ELEADIGDIT ALEKYLALSQ YGNILALEEL 
               
               
                   
                   
               
               
                   
                 NKALEELDED VGWNQNDIAN LEDDVETLTK NQNAFAEQGE 
               
               
                   
                   
               
               
                   
                 AIKEDLQGLA DFVEGQEGKI LQNETSIKKN TQRNLVNGFE 
               
               
                   
                   
               
               
                   
                 IEKNKDAIAK NNESIEDLYD FGHEVAESIG EIHAHNEAQN 
               
               
                   
                   
               
               
                   
                 ETLKGLITNS IENTNNITKN KADIQALENN VVEELFNLSG 
               
               
                   
                   
               
               
                   
                 RLIDQKADID NNINNIYELA QQQDQHSSDI KTLKKNVEEG 
               
               
                   
                   
               
               
                   
                 LLELSDHIID QKTDIAQNQA NIQDLATYNE LQDQYAQKQT 
               
               
                   
                   
               
               
                   
                 EAIDALNKAS SENTQNIEDL AAYNELQDAY AKQQTEAIDA 
               
               
                   
                   
               
               
                   
                 LNKASSENTQ NIEDLAAYNE LQDAYAKQQA EAIDALNKAS 
               
               
                   
                   
               
               
                   
                 SENTQNIAKN QADIANNITN LYELAQQQDK 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  125 Iodine 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 UspA1 (conserved fragments from the fibro- 
               
               
                 nectin binding domain - ‘/’ separating alternative 
               
               
                 choices of an amino acid at a position) 
               
               
                 (SEQ ID NO: 34) 
               
               
                 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 V G A G Q/N/E/K I S/R A/D T/D S T D A V N G S 
               
               
                   
               
               
                 Q L H/Y A L A S/K/T T/A/V I/V 
               
               
                   
               
               
                 (SEQ ID NO: 35) 
               
               
                 S T D A V N G S Q L 
               
               
                   
               
               
                 (SEQ ID NO: 36) 
               
               
                 L L N/D L S G R L L/I D Q K A D I D N N I N N/H I 
               
               
                   
               
               
                 Y E/D L A Q Q Q D Q H S S D I K T L K 
               
               
                   
               
               
                 (SEQ ID NO: 37) 
               
               
                 D Q K A D I D N N I N 
               
               
                   
               
               
                 (SEQ ID NO: 38) 
               
               
                 L A Q Q Q D Q H S S D I K T L K 
               
               
                   
               
               
                 From UspA2 (conserved fragments from the fibro- 
               
               
                 nectin binding domain - ‘/’ separating alternative 
               
               
                 choices of an amino acid at a position) 
               
               
                 (SEQ ID NO: 39) 
               
               
                 K A D I D N N I N N/H I Y E L A Q Q Q D Q H S S D 
               
               
                   
               
               
                 (SEQ ID NO: 40) 
               
               
                 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 - ‘/’ separating alternative 
               
               
                 choices of an amino acid at a position) 
               
               
                 (SEQ ID NO: 41) 
               
               
                 I E/Q D L A A Y N E L Q D A Y A K Q Q A/T E A I D 
               
               
                   
               
               
                 A L N K A S S E N T Q N I A K N Q A D I A N N I 
               
               
                   
               
               
                 T/N N I Y E L A Q Q Q D K/Q H R/S S D I K T L A K 
               
               
                   
               
               
                 T/A S A A N T D/N R I 
               
               
                   
               
               
                 (SEQ ID NO: 42) 
               
               
                 D L A A Y N E L Q D A Y A K Q Q 
               
               
                   
               
               
                 (SEQ ID NO: 43) 
               
               
                 E A I D A L N K A S S E N T Q N I A K N Q A D I A 
               
               
                   
               
               
                 N N I 
               
            
           
         
       
     
     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 domains 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  UspA1 or UspA2 proteins by truncation at either or both of the N- and C-termini. Truncates are not the full-length native UspA1 or A2 molecules. Accordingly, the invention further provides a wild-type UspA1 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). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Possible combinations of truncations to the N- and C- termini of wild-type UspA1 protein. 
               
               
                 No. of amino acids lacking, at least or exactly:  
               
            
           
           
               
               
            
               
                 From the 
                   
               
               
                 N-terminus 
                 From the C-terminus 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 0 
                 X 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100  
                 120 
                 140  
                 160 
                 180 
                 200 
                 220  
                 240 
                 260  
                 280 
                 300 
                 320 
                 340 
                 360  
                 380 
                 400 
                 420  
                 440 
                 450 
               
               
                 20 
                 0 
                 20 
                 30 
                 40  
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180  
                 200 
                 220  
                 240  
                 260  
                 280 
                 300 
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 30 
                 0  
                 20 
                 30 
                 40  
                 50 
                 60 
                 70  
                 80 
                 100  
                 120 
                 140  
                 160 
                 180 
                 200 
                 220  
                 240  
                 260  
                 280 
                 300 
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 40 
                 0 
                 20 
                 30 
                 40  
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180  
                 200 
                 220  
                 240 
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 50 
                 0 
                 20 
                 30 
                 40  
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180  
                 200 
                 220  
                 240 
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 60 
                 0 
                 20 
                 30 
                 40  
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180  
                 200 
                 220  
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 70 
                 0  
                 20 
                 30 
                 40  
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180  
                 200 
                 220  
                 240 
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 80 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180 
                 200 
                 220  
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 100 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100  
                 120  
                 140  
                 160 
                 180 
                 200 
                 220  
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 120 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180  
                 200 
                 220  
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 140 
                 0  
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180 
                 200 
                 220  
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 160 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180 
                 200 
                 220  
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 180 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80 
                 100  
                 120  
                 140  
                 160 
                 180 
                 200 
                 220  
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 200 
                 0  
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80 
                 100 
                 120 
                 140  
                 160 
                 180  
                 200 
                 220 
                 240  
                 260 
                 280 
                 300  
                 320 
                 240 
                 360 
                 380  
                 400 
                 420 
                 440 
                 450 
               
               
                 220 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80  
                 100 
                 120  
                 140  
                 160 
                 180 
                 200  
                 220 
                 240 
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 240 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80  
                 100 
                 120  
                 140  
                 160 
                 180  
                 200  
                 220 
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 260 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80  
                 100 
                 120  
                 140  
                 160 
                 180  
                 200  
                 220 
                 240 
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 280 
                 0 
                 20  
                 30 
                 40 
                 50 
                 60 
                 70  
                 80  
                 100 
                 120  
                 140  
                 160 
                 180  
                 200 
                 220 
                 240  
                 260  
                 280 
                 300  
                 320  
                 240 
                 360  
                 380  
                 400 
                 420  
                 440 
                 450 
               
               
                 298 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70  
                 80 
                 100 
                 120  
                 140 
                 160 
                 180  
                 200  
                 220 
                 240 
                 260 
                 280 
                 300  
                 320 
                 240 
                 360 
                 380  
                 400 
                 420 
                 440 
                 450 
               
               
                   
               
            
           
         
       
     
     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. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Possible combinations of truncations to the N- and C- termini of wild-type UspA2 protein 
               
               
                 No. of amino acids lacking, at least or exactly 
               
            
           
           
               
               
            
               
                 From the 
                   
               
               
                 N-terminus 
                 From the C-terminus 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 0 
                 X 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 20 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 30 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 40 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 50 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 60 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 70 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 80 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 100 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 120 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 140 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 160 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                 164 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 180 
                 200 
                 220 
                 240 
                 260 
                 280 
                 300 
                 312 
               
               
                   
               
            
           
         
       
     
     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 C-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. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Possible combinations of truncations to the N- and 
               
               
                 C- termini of wild-type UspA2 protein 
               
               
                 No. of amino acids lacking, at least or exactly: 
               
            
           
           
               
               
            
               
                 From the 
                   
               
               
                 C-terminus 
                 From the N-terminus 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 0 
                 X 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 20 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 30 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 40 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 50 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 60 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 70 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 80 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 100 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 120 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 140 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 160 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 180 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 200 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 220 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 240 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 260 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 280 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 300 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 320 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 340 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 360 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 380 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 400 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 420 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 440 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                 453 
                 0 
                 5 
                 10 
                 15 
                 20 
                 25 
                 29 
               
               
                   
               
            
           
         
       
     
     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 
               
               
                 No. of amino acids lacking, at least or exactly: 
               
            
           
           
               
               
            
               
                 From the  
                   
               
               
                 N-terminus 
                 From the C-terminus 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 0 
                 X 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 172 
               
               
                 20 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 172 
               
               
                 30 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 172 
               
               
                 40 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 172 
               
               
                 50 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 172 
               
               
                 60 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 172 
               
               
                 70 
                 0 
                 20 
                 30 
                 40 
                 50 
                 60 
                 70 
                 80 
                 100 
                 120 
                 140 
                 160 
                 172 
               
               
                 80 
                 0 
                 20 
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     Known wild-type UspA1 sequences that may be truncated in this way are those of strains ATCC25238 (MX2; GenBank accession no. AAD43465), P44 (AAN84895), 035E (AAB96359), TTA37 (AAF40122), O12E (AAF40118), O46E (AAF36416), V1171 (AAD43469), TTA24 (AAD43467) (see Table 1/ FIGS. 19A through 19D ); or BC5 (see above). Known wild-type UspA2 sequences that may be truncated in this way are those of strains O35E (GenBank accession no. 04407), 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/ FIGS. 20A through 20J ); or BC5 [Forsgren_UspA2] (see above). 
     Ideally the UspA1 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 UspA1 (conserved fragments from the fibro- 
               
               
                 nectin binding domain - ‘/’ separating alternative 
               
               
                 choices of an amino acid at a position) 
               
               
                 (SEQ ID NO: 44) 
               
               
                 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 V G A G Q/N/E/K I S/R A/D T/D S T D A V N G S 
               
               
                   
               
               
                 Q L H/Y A L A S/K/T T/A/V I/V 
               
               
                   
               
               
                 (SEQ ID NO: 45) 
               
               
                 S T D A V N G S Q L 
               
               
                   
               
               
                 (SEQ ID NO: 46) 
               
               
                 L L N/D L S G R L L/I D Q K A D I D N N I N N/H I 
               
               
                   
               
               
                 Y E/D L A Q Q Q D Q H S S D I K T L K 
               
               
                   
               
               
                 (SEQ ID NO: 47) 
               
               
                 D Q K A D I D N N I N 
               
               
                   
               
               
                 (SEQ ID NO: 48) 
               
               
                 L A Q Q Q D Q H S S D I K T L K 
               
               
                   
               
               
                 From UspA2 (conserved fragments from the fibro- 
               
               
                 nectin binding domain - ‘/’ separating alternative 
               
               
                 choices of an amino acid at a position) 
               
               
                 (SEQ ID NO: 49) 
               
               
                 K A D I D N N I N N/H I Y E L A Q Q Q D Q H S S D 
               
               
                   
               
               
                 (SEQ ID NO: 50) 
               
               
                 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 - ‘/’ separating alternative 
               
               
                 choices of an amino acid at a position) 
               
               
                 (SEQ ID NO: 51) 
               
               
                 I E/Q D L A A Y N E L Q D A Y A K Q Q A/T E A I D 
               
               
                   
               
               
                 A L N K A S S E N T Q N I A K N Q A D I A N N I 
               
               
                   
               
               
                 T/N N I Y E L A Q Q Q D K/Q H R/S S D I K T L A K 
               
               
                   
               
               
                 T/A S A A N T D/N R I 
               
               
                   
               
               
                 (SEQ ID NO: 52) 
               
               
                 D L A A Y N E L Q D A Y A K Q Q 
               
               
                   
               
               
                 (SEQ ID NO: 53) 
               
               
                 E A I D A L N K A S S E N T Q N I A K N Q A D I A 
               
               
                   
               
               
                 N N I 
               
            
           
         
       
     
     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  lipooligosaccaharides, 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. 
       FIGS. 19A through 19D and 20A through 20J  show an alignment of peptide sequences of UspA1 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  FIGS. 19A through 19D and 20A through 20J . 
     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 (UspA1 and/or UspA2) can provide an immunogenic composition giving the host enhanced defensive capabilities against Moraxella&#39;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 UspA1 and/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 
         FIGS. 1A through 1I  show thirteen  M. catarrhalis  strains tested for fibronectin binding ( FIG. 1A ). Strong fibronectin binding correlated to UspA1/A2 expression as detected by anti-UspA1/A2 pAb ( FIG. 1B  (wild type)  FIG. 1C  (ΔuspA1),  FIG. 1D  (ΔuspA2),  FIG. 1E  (ΔuspA1/A2),  FIG. 1F  (wild type),  FIG. 1G  (ΔuspA1),  FIG. 1H  (ΔuspA2),  FIG. 1I  (ΔuspA1/A2)). Flow cytometry profiles of  M. catarrhalis  BBH18 wild type and UspA1/A2 deficient mutants show an UspA1/A2-dependent binding to soluble fibronectin. The profiles of wild type clinical isolate ( FIGS. 1B and 1F ) and corresponding mutants devoid of UspA1 ( FIG. 1C  and  FIG. 1G ), or UspA2 ( FIG. 1D  and  FIG. 1H ), and double mutants ( FIG. 1E  and  FIG. 1I ) lacking both UspA1 and UspA2 are shown. Bacteria were incubated with rabbit anti-UspA1/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. 
         FIG. 2  shows that  M. catarrhalis  RH4 UspA2 deficient mutants do not bind  125 I-labeled fibronectin.  E. coli  BL21 was included as a negative control not binding fibronectin. Bacteria were incubated with  125 I-labeled fibronectin followed by several washes and analyzed in a gamma counter. Fibronectin binding to the RH4 wild type expressing both UspA1 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. 
         FIGS. 3A through 3D  show pictures that verify that  M. catarrhalis  mutants devoid of UspA1 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 ( FIG. 3A ).  M. catarrhalis  ΔuspA1 mutant was also retained at a high density ( FIG. 3B ), whereas  M. catarrhalis  ΔuspA2 and ΔuspA1/A2 double mutants adhered poorly ( FIG. 3C  and  FIG. 3D ). Glass slides were coated with fibronectin and incubated with  M. catarrhalis  RH4 and its corresponding UspA1/A2 mutants. After several washes, bacteria were Gram stained. 
         FIG. 4  is a graph showing that recombinant UspA1 and A2 bind to fibronectin in a dose-dependent manner. Specific fibronectin binding is shown for UspA1 50-770  and UspA2 30-539 . 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. 
         FIGS. 5A and 5B . The active fibronectin binding domains for UspA1 and UspA2 are located between amino acids 299 to 452 and 165 to 318, respectively. Truncated proteins derived from UspA1 ( FIG. 5A ) and UspA2 ( FIG. 5B ) 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 μg/ml and 120 μg/ml fibronectin for UspA1 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. 
         FIG. 6  shows the sequence according to sequence ID No. 1, and the sequence homology between UspA1 300-453  (SEQ ID NO: 87) and UspA2 165-318  (SEQ ID NO: 3). The 31 identical amino acid residues are within brackets. 
         FIG. 7  shows that truncated UspA1 50-491  and UspA1 299-452  fragments competitively inhibit  M. catarrhalis  UspA-dependent fibronectin binding.  M. catarrhalis  ΔuspA1/A2 double mutants, which do not bind fibronectin, were included as negative controls. UspA1 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. UspA1 50-491  and UspA1 299-452  resulted in 95% and 63% inhibition respectively. Error bars represent mean±SD of three independent experiments. 
         FIGS. 8A through 8B  show that UspA1 299-452  and UspA2 165-318  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 ( FIG. 8A ). Pre-incubation with the fibronectin binding proteins UspA1 299-452 , UspA2 165-318 , or anti-fibronectin pAb resulted in significantly reduced binding by  M. catarrhalis  RH4 as compared to control recombinant proteins (UspA1 433-580  and UspA2 30-177 ) and a control antibody (anti-ICAM1 mAb) ( FIG. 8B ). P&lt;0.05 by two-tailed paired Student&#39;s t test. Mean values of three separate experiments are shown and error bars indicate SD. 
         FIGS. 9A through 9B  show binding of  M. catarrhalis  RH4 to laminin via UspA1 and A2.  M. catarrhalis  RH4 wild type (wt) strongly bound to immobilized laminin with a mean OD of 1.27. RH4ΔuspA1 showed mean OD of 1.14 (89.8% of the wild type). RH4ΔuspA2 and the double mutant RH4ΔuspA1/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 μg/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 UspA1 and A2 laminin in a dose-dependent manner. Specific laminin binding is shown for UspA1 50-770  and UspA2 30-539 . 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. 
         FIGS. 10A through 10B  show that the active laminin binding domains for UspA1 50-770  ( FIG. 10A ) and UspA2 30-539  ( FIG. 10B ) are located in the N-terminal halves. Forty nM of recombinant UspA1 50-770  and UspA2 30-539  together with the truncated proteins were coated on microtiter plates and incubated with 20 μg/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. 
         FIGS. 11A through 11C  is a schematic illustration of C3, covalent bound C3b and C3met. ( FIG. 11A ) The C3-molecule in serum consists of one α-chain and one β-chain. ( FIG. 11B ) The α-chain contains an internal thioester site that after activation can attach covalently to a microbial surface. ( FIG. 11C ) The C3 has been treated with methylamine, which becomes covalently attached to the thioester. 
         FIGS. 12A through 12B  illustrates that  M. catarrhalis  counteracts the classical and alternative pathways of the complement system by the outer membrane proteins UspA1 and A2. ( FIG. 12A )  M. catarrhalis  RH4 wild-type (wt), the ΔuspA1, the ΔuspA2 or the ΔuspA1/A2 mutants were incubated in the presence of 10% NHS. ( FIG. 12B ) The ΔuspA1/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. ( FIG. 12A ) The mean values after 5 min for the ΔuspA1, the ΔuspA2 or the ΔuspA1/A2 mutants were significantly different from the wild-type (P&lt;0.05). ( FIG. 12B ) The mean values after 5 min for the ΔuspA1/A2 mutant and after 10 min for the ΔuspA1/A2 mutant incubated Mg-EGTA were significantly different from the wild-type (P&lt;0.05). 
         FIGS. 13A through 13B  illustrates that  Moraxella catarrhalis  binds C3 in serum independently of complement activation. Flow cytometry profiles showing C3 binding to ( FIG. 13A )  M. catarrhalis  RH4 or ( FIG. 13B )  Streptococcus pneumoniae . Bacteria were incubated with NHS or NHS pretreated with EDTA. Thereafter, a rabbit anti-human C3d pAb and as a secondary 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. 
         FIGS. 14A through 14C  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 ( FIG. 14A ) binding with increasing concentrations of C3met.  FIG. 14B  shows the mean fluorescence intensity (mfi) of each profile in panel  FIG. 14A .  FIG. 14C  shows that 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  FIG. 3 . Error bars indicate SD. *P≤0.05, **P≤0.014, ***P≤0.001. 
         FIGS. 15A through 15O  illustrates that flow cytometry profiles of  M. catarrhalis  RH4 wild type and UspA1/A2 deficient mutants show a UspA1/UspA2-dependent C3met/C3 binding. The profiles of a wild type clinical isolate ( FIG. 15A ,  FIG. 15F ,  FIG. 15K ) and corresponding mutants devoid of protein MID ( FIG. 15B ,  FIG. 15G ,  FIG. 15L ), UspA1 ( FIG. 15C ,  FIG. 15H ,  FIG. 15M ), UspA2 ( FIG. 15D ,  FIG. 15I ,  FIG. 15N ), or both UspA1 and UspA2 ( FIG. 15E ,  FIG. 15J ,  FIG. 15O ) are shown. Bacteria were incubated with C3met ( FIGS. 15A through 15E ). NHS-EDTA ( FIGS. 15F through 15J ) or NHS ( FIGS. 15K through 15O ) and detected as outlined in  FIG. 3 . One typical experiment out of three with the mean fluorescence intensity (mfi) for each profile is shown. 
         FIGS. 16A through 16B  illustrates that C3met binds to purified recombinant UspA2 30-539 , whereas only a weak C3met binding to UspA1 50-770  is observed. Furthermore, the C3met binding region of UspA2 was determined to be located between the amino acid residues 200 to 458. In  FIG. 16A , the recombinant UspA1 50-770  and UspA2 30-539  were immobilized on a nitrocellulose membrane. The membrane was incubated with [ 125 I]-labelled C3met overnight and bound protein was visualized with a Personal FX (Bio-Rad) using intensifying screens. The recombinant protein MID 962-1200  was included as a negative control. In  FIG. 16B , UspA1 50-770 , UspA2 30-539  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. 
         FIGS. 17A through 17C  illustrates that addition of recombinant UspA1 50-770  and UspA2 30-539  to serum inhibit C3b deposition and killing of  M. catarrhalis  via the alternative pathway. Flow cytometry profiles show C3b-deposition on RH4ΔuspA1/A2 after incubation with ( FIG. 17A ) NHS or NHS preincubated with recombinant (rec.) UspA1 50-770  and UspA2 30-539 , or ( FIG. 17B ) NHS-Mg-EGTA or NHS-Mg-EGTA preincubated with UspA1 50-770  and UspA2 30-539 . After addition of the various NHS combinations, bacteria were analyzed as described in  FIG. 13 . RH4ΔuspA1/A2 ( FIG. 17C ) was incubated with 10% NHS or NHS-Mg-EGTA. For inhibition, the NHS-Mg-EGTA was incubated with 100 nM UspA1 50-770  and/or UspA2 30-539  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 ΔuspA1/A2 mutant preincubated with recombinant proteins were significantly different from the ΔuspA1/A2 mutant incubated with Mg-EGTA alone (P&lt;0.05). 
         FIG. 18  illustrates that recombinant UspA1 50-770  and UspA2 30-539  decrease haemolysis of rabbit erythrocytes by inhibition of the alternative pathway. NHS was incubated with or without 100 nM UspA1 50-770  and/or UspA2 30-539  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+UspA2 30-539  and NHS+UspA1 50-770 /UspA2 30-539  at NHS concentrations of 2, 3 and 4% were significantly different from the NHS control (P&lt;0.05). 
         FIGS. 19A through 19D  illustrate a pileup-analysis of UspA1 for eight different strains, to show the homology of different parts of UspA1 (SEQ ID NOS 11-18 are disclosed respectively in order of appearance). Each page, and hence each of  FIGS. 19A through 19D , represents a different portion of the alignment, which was too large to fit in one page. 
         FIGS. 20A through 20J  illustrate a pileup-analysis of UsPa2 for thirteen different strains to show the homology of different parts of UspA2 (SEQ ID NOS 19-31 are disclosed respectively in order of appearance). Each page, and hence each of  FIGS. 20A through 20J , represents a different portion of the alignment, which was too large to fit in one page. 
         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 UspA1-deficient mutants were cultured in BHI supplemented with 1.5 μg/ml chloramphenicol (Sigma, St. Louis, Mo.), and UspA2-deficient mutants were incubated with 7 μg/ml zeocin (Invitrogen, Carlsbad, Calif.). 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]           D1   Sputum   [53]           Ri49   Sputum   [53]           C10   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]           SÖ-1914   Tympanic cavity aspirate   [23]               Note:       The strains C10, R4 did not have the uspA1 gene, whereas F16, R14, Z14 lacked the uspA2 gene. [10] The remaining strains contained both uspA1 and A2 genes (data not shown).            
DNA Method
 
     To detect the presence uspA1, A2, 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 UspA1 299-452  and UspA2 165-318  5′ and 3′ primers of the respective uspA1 and uspA2 gene of RH4 and BBH18. Confirmation of the presence of the amino acid residues “DQKADIDNNINNIYELAQQQDQHSSDIKTLK” (SEQ ID NO: 1) was also performed by PCR with a primer (5′-CAAAGCTGACATCCAAGCACTTG-3′) (SEQ ID NO: 54) designed from the 5′ end of this sequence and 3′ primers for uspA1 and A2 as described by Meier at al. [50] 
     Recombinant Proteins Construction and Expression 
     Recombinant UspA1 50-770  and UspA 230-539 , 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 UspA1 50-770  and UspA2 30-539  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&#39;s instructions for native conditions. The recombinant proteins were analyzed on SDS-PAGE as described.[21] 
                     TABLE 8                  Primers used in this present study (5′ primers are       disclosed as SEQ ID NOS 55-69, respectively, in       order of appearance; 3′ primers are disclosed as       SEQ ID NOS 70-84, respectively, in order of       appearance.                     Protein   5′ primer                   3′ primer               UspA1 50-770     gcgtctgcggatccagtaggcaaggcaacc           ccctgaagctttagtgcataacctaattg               UspA1 50-491     gcgtctgcggatccagtaggcaaggcaacc           ttgagcaagcttagcttggtttttagcg               UspA1 50-197     gcgtctgcggatccagtaggcaaggcaacc           acctgtggcaagcttcttcctgcc               UspA1 50-321     gcgtctgcggatccagtaggcaaggcaacc           ggtgtcactaagcttacctgcaccaacatgaac               UspA1 299-452     ggatttgcaggtgcatcggatcctggtaatggtact           gtcttttgtaagatcaagcttttgatcaat               UspA1 433-580     catagctctgatatggatccacttaaaaac           catgctgagaagcttacctagattgg               UspA1 557-704     gccaaagcacaagcggatccaaataaagac           ggtcttattggtagtaagcttagcttggttttg               UspA1 680-770     gttgagcaaaaggatcccatcaatcaagag           ccctgaagctttagtgcataacctaattg               UspA2 30-539     cgaatgcggatcctaaaaatgatataactttagagg           cattaagcttggtgtctaatgcagttac               UspA2 30-177     cgaatgcggatcctaaaaatgatataactttagagg           ctcatgaccaaaatcaagcttatcttcgatagactc               UspA2 101-240     gatattgcggatccggaagatgatgttgaaac           gatcaataagcttaccgcttagattgaatagttcttc               UspA2 101-315     gatattgcggatccggaagatgatgttgaaac           gtcaatcgcttcaagcttcttttgagcatactg               UspA2 165-318     gagattgagaaggatccagatgctattgct           gtcaatcgcttcaagcttcttttgagcatactg               UspA2 302-458     gctcaaaaccaagcggatccccaagatctg           ggtgagcgtttcaagctttgcatcagcatcggc               UspA2 446-539     gcaagtgctgcggatcctgatcgtattgct           cattaagcttggtgtctaatgcagttac                    
Antibodies
 
     Rabbit anti-UspA1/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 UspA1/A2-protein expression and the capacity of  M. catarrhalis  to bind fibronectin were analyzed by flow cytometry.  M. catarrhalis  wild type strains and UspA1/A2-deficient mutants were grown overnight and washed twice in phosphate buffered saline containing 3% fish gelatin (PBS-gelatin). The bacteria (10 8 ) were then incubated with the anti-UspA1/A2 antiserum or 5 μg 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&#39;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, Fla.). All incubations were kept in a final volume of 100 μl 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 μmoles of UspA fragments for 1 h with 2 μg of fibronectin before incubation with  M. catarrhalis  bacteria (10 8 ). 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 μl 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  125 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 (10 8 ) were incubated for 1 h at 37° C. with  125 I-labeled fibronectin (1600 kcpm/sample) in PBS containing 2% BSA. After three washings with PBS 2% BSA,  125 I-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 UspA1 50-770  and UspA2 30-539  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 μg/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 OD 450 . ELISAs with truncated proteins spanning UspA1 50-770  and UspA2 30-539  were performed with fixed doses of fibronectin at 80 μg/ml and 120 μg/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 μg 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 10 4  cells/well in 200 μl of gentamicin-free culture medium. Thereafter, cells were incubated overnight at 37° C. in a humidified atmosphere of 5% CO 2  and 95% air. On the day of experiments, inhibition of  M. catarrhalis  adhesion was carried out by pre-incubating increasing concentration of recombinant UspA1/A2 truncated proteins containing the fibronectin binding domains (UspA1 299-452  and UspA2 165-318 ) or rabbit anti-human fibronectin pAb (diluted 1:50) for 1 h. Nonfibronectin binding recombinant proteins (UspA1 433-580  and UspA2 30-177 ) were used as controls. Chang epithelial cells are known to express ICAM1.[18] Hence an anti-ICAM1 antibody was used to differentiate if the inhibitory effect of the anti-fibronectin antibody was secondary to steric hindrance. Subsequently,  M. catarrhalis  RH4 (10 6 ) in PBS-gelatin was inoculated onto the confluent monolayers. In all experiments, tissue culture plates were centrifuged at 3,000×g for 5 min and incubated at 37° C. in 5% CO 2 . 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% CO 2 . 
     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 (1×10 6 /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 UspA1.[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 UspA1-deficient, UspA2-deficient and double mutants were cultured in BHI supplemented with antibiotics as described.[58] 
     Recombinant Protein Construction and Expression 
     Recombinant UspA1 50-770  and UspA2 30-539 , which are devoid of their hydrophobic C-termini, were manufactured.[58] In addition, recombinant proteins corresponding to multiple regions spanning UspA1 50-770  and UspA2 30-539  were used. [78] 
     Antibodies 
     Rabbit anti-UspA1/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 μg/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 (10 8 ) in 100 μl 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 OD 450  according to a standard protocol. 
     Enzyme-Linked Immunosorbent Assay (ELISA) 
     Microtiter plates (Nunc-Immuno Module) were coated with 40 nM of purified recombinant UspA1 50-770  and UspA2 30-539  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 NaCl, 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 OD 450 . Uncoated wells incubated with identical dilutions of laminin were used as background controls. ELISAs with truncated proteins spanning UspA1 50-770  and UspA2 30-539  were performed with fixed doses of laminin (20 μg/ml). 
     Interaction between  M. 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, Malmö University Hospital, Lund University, Sweden. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                   M. catarrhalis  is a unique C3/C3met binding 
               
               
                 bacterium. Related  moraxella  subspecies and other common 
               
               
                 human pathogens do not bind C3/C3met (mfi &lt; 2.0). After 
               
               
                 incubation with EDTA-treated NHS or C3met, bacteria were 
               
               
                 analysed by flow cytometry using a rabbit anti-C3d pAb and a 
               
               
                 FITC-conjugated goat anti-rabbit pAb. 
               
            
           
           
               
               
               
            
               
                 Species 
                 NHS-EDTA (mfi) 
                 C3met (mfi) 
               
               
                   
               
            
           
           
               
               
               
            
               
                   Moraxella catarrhalis  RH4 
                 8.7 
                 22.1 
               
               
                 
                   M. osloensis 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   M. bovis 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   M. caniculi 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   M. nonliquefacie 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   N. pharyngis 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   N. sicca 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   N. flava 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   N. subflava 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   Oligella ureolytica  (n = 2) 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   Haemophilus influenzae  (n = 7) 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   Streptococcus pneumoniae  (n = 11) 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   Legionella pneumophila  (n = 2) 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   Pseudomonas aeruginosa  (n = 2) 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   Listeria monocytogenes 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   Yersinia entercolitica 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   Staphylococcus aureus  (n = 3) 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   Streptococcus pyogenes  (n = 2) 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   Streptococcus agalactia 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   Enterococcus faecalis 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   Helicobacter pylori 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   Escherichia coli  (n = 2) 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   M. ovis 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   M. caviae 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   Neisseria gonorrheae 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   N. meningtidis 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                 
                   N. mucosa 
                 
                 &lt;2.0 
                 &lt;2.0 
               
               
                   
               
            
           
         
       
     
     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 μg/ml kanamycin. The UspA1-deficient mutants were cultured in BHI supplemented with 1.5 μg/ml chloramphenicol (Sigma, St. Louis, Mo.), and UspA2-deficient mutants were incubated with 7 μg/ml zeocin (Invitrogen, Carlsbad, Calif.). Both chloramphenicol and zeocin were used for growth of the UspA1/A2 double mutants. 
     Antibodies 
     Rabbits were immunized intramuscularly with 200 μg recombinant full-length UspA1 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-UspA1 antiserum was affinity-purified with Sepharose-conjugated recombinant UspA1 50-770 .[58] The antiserum bound equally to UspA1 and UspA2 and was thus designated anti-UspA1/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, Calif.). The horseradish peroxidase (HRP)-conjugated donkey anti-goat pAb was obtained from Serotec (Oxford, UK). 
     Proteins and Iodine Labelling 
     The manufacture of recombinant UspA1 50 770  and UspA2 30 539 , which are devoid of their hydrophobic C-termini, has recently been described.[23] The truncated UspA1 and UspA2 proteins were manufactured as described in detail by Tan et al.[78] C3b was purchased from Advanced Research Technologies. C3(H 2 O) 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  125 I (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 (10 8  colony forming units; cfu) were incubated with C3met, C3b, C3(H 2 O), or 10% NHS with or without 10 mM EDTA or 4 mM MgCl 2  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, Fla.). All incubations were kept in a final volume of 100 μl 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 UspA1 50-770  and UspA2 30-539  proteins for 30 min at 37° C. To analyze the characteristics of the  M. catarrhalis  and C3 interaction, increasing concentrations of NaCl (0-1.0 M) was added to bacteria and C3met. To analyze UspA1/A2 expression, bacteria (10 8  cfu) were incubated with the anti-UspA1/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 UspA1 and UspA2,  M. catarrhalis  was incubated with or without EDTA followed by detection of UspA1/A2 expression. EDTA, at the concentrations used in the NHS-EDTA experiments, did not change the density of UspA1/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 □-chain of C4BP.[41] The flow through was collected and the depleted serum was stored in aliquots at −70° C. Serum depleted of C1q was obtained via the first step of C1q purification [79] using Biorex 70 ion exchange chromatography (Bio-Rad, Hercules, Calif.). The resulting sera displayed normal haemolytic activity. The factor D and properdin deficient serum was kindly provided by Dr. Anders Sjöholm (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 MgCl 2 , 0.15 mM CaCl 2 , and 2.5% dextrose (DGVB ++ ). Bacteria (10 3  cfu) were incubated together with 10% NHS and EDTA or Mg-EGTA in a final volume of 100 μl. The bacteria/NHS was incubated at 37° C. and at various time points, 10 μl aliquots were removed and spread onto BHI agar plates. In inhibition studies, 10% serum was incubated with 100 nM of the recombinant UspA1 50-770  and UspA2 30-539  proteins for 30 min at 37° C. before bacteria were added. 
     Dot Blot Assays 
     Purified recombinant UspA1 50-770  and UspA2 30-539  diluted in three-fold steps (1.9-150 nM) in 100 μl of 0.1 M Tris-HCl, pH 9.0 were applied to nitrocellulose membranes (Schleicher &amp; Schüll, 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 [ 125 I]-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 UspA1 50-770  or UspA2 30-539  and C3 was further analysed using surface plasmon resonance (Biacore 2000; Biacore, Uppsala, Sweden) as recently described for the UspA1/2-C4BP interaction.[58] The K D  (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 UspA1 50-770 , UspA2 30-539 , or the truncated UspA1 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 □g 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 OD 450 . 
     Haemolytic 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 MgCl 2 , 10 mM EGTA, and 2.5% dextrose (Mg ++ EGTA), and resuspended at a concentration of 0.5×10 9  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 spectrophotometric measurement of released hemoglobin at 405 nm. For inhibition with UspA1 and UspA2, 10% serum was preincubated with 100 nM of recombinant UspA1 50-770  and/or UspA2 3C-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 (Pharmalink AB, Upplands Väsby, Sweden). The PMN were centrifuged for 10 min at 300 g, washed in PBS and resuspended in RPMI 1640 medium (Life Technologies, Paisley, Scotland). The bacterial suspension (0.5×10 8 ) was opsonized with 3% of either NHS or NHS-EDTA, or 20 □g of purified C3met for 15 min at 37° C. After washes, bacteria were mixed with PMN (1×10 7  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 UspA1 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 UspA1/A2 expression by flow cytometry analysis. High UspA1/A2 expression as determined by high mean fluorescence intensity (MFI) was correlated to UspA1/A2 expression (Pearson correlation coefficient 0.77, P&lt;0.05) ( FIG. 1A ). However, to discriminate between UspA1 and A2 expression was not possible with our anti-UspA1/A2 pAb. Moreover, the presence of UspA2H protein contributing to the binding was unlikely as the uspA2H gene was not found in the strains used in this study (data not shown). 
     Two  M. catarrhalis  isolates (BBH18 and RH4) and their specific mutants lacking UspA1, 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 ( FIG. 1F ). In contrast, BBH18ΔuspA1 showed a decreased fibronectin binding with an MFI of 68.6 ( FIG. 1G ). Fibronectin binding to BBH18ΔuspA2 and the double mutant BBH18ΔuspA1/A2 revealed an MFI of only 10.7 and 11.5, respectively ( FIG. 1H, 1I ). Similar results were obtained with UspA1/A2 mutants of the clinical strain  M. catarrhalis  RH4. Taken together, these results suggest that UspA1 and A2 bound fibronectin and that the ability of the bacteria to bind fibronectin strongly depended on UspA1/A2 expression. 
     To further analyze the interaction between fibronectin and  M. catarrhalis,    125 I-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  125 I-fibronectin while the corresponding ΔuspA1 mutant showed 80% binding of the wild type. In contrast, the ΔuspA2 and double mutant bound  125 I-fibronectin at 14% and 12%, respectively, which was just above the background levels (5.0 to 10%) ( FIG. 2 ). Similar results were obtained with  M. catarrhalis  BBH18 and the corresponding UspA1/A2 mutants. Thus, our results suggest that both UspA1 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 ΔuspA1/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 ΔuspA1 mutant were found to strongly adhere to the fibronectin coated glass slides ( FIGS. 3A and 3B ). In contrast,  M. catarrhalis  ΔuspA2 and ΔuspA1/A2 double mutants weakly adhered to the fibronectin coated glass slide with only a few bacteria left after washing ( FIGS. 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  M. catarrhalis  binding to immobilized fibronectin. 
     The Fibronectin Binding Domains include Amino Acid Residues Located between 299 and 452 of UspA1 and between 165 and 318 of UspA2 
     To further analyze the interactions of UspA1 and A2 with fibronectin, truncated UspA1 50-770  and UspA2 30-539  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 UspA1 50-770  and UspA2 30-539  bound soluble fibronectin and the interactions were dose-dependent ( FIG. 4 ). 
     To define the fibronectin-binding domain of UspA1, recombinant proteins spanning the entire molecule of UspA1 50-770  were manufactured. Fibronectin was incubated with the immobilized UspA1 proteins fragments and the interactions were quantified by ELISA. UspA1 50-491  bound fibronectin almost as efficiently as UspA1 50-770  suggesting that the binding domain was within this part of the protein. Among the other truncated fragments, UspA1 299-452  efficiently bound fibronectin ( FIG. 5A ). In parallel, the interactions between fibronectin and several recombinant UspA2 fragments including amino acids UspA2 30-539  were analyzed. The two fragments UspA2 101 318  and UspA2 165 318  strongly bound fibronectin ( FIG. 5B ). Our findings provide significant evidence that the binding domains include residues found within UspA1 299-452  and UspA2 165-318 . A sequence comparison between these two binding fragments revealed that the 31 amino acid residues “DQKADIDNNINNIYELAQQQDQHSSDIKTLK” (SEQ ID NO: 1) were identical for UspA1 and A2 ( FIG. 6 ). Moreover, this repeat sequence was also found in the uspA1 and A2 gene of  M. catarrhalis  BBH18 and RH4 (data not shown). 
     UspA1 5C-491  and UspA1 299-452  Fragments Competitively Inhibit  M. catarrhalis  Fibronectin Binding 
     To further validate our findings on the UspA1/A2 fibronectin binding domains, recombinant truncated UspA1 proteins were tested for their capacity to block fibronectin binding to  M. catarrhalis . Fibronectin (2 μg) was pre-incubated with 0.25 μmoles of recombinant UspA1 fragments and subsequently incubated with  M. catarrhalis . Finally,  M. catarrhalis  UspA-dependent fibronectin binding was measured by flow cytometry. Pre-incubation with UspA1 50-491  and UspA1 299-452  resulted in decreased fibronectin binding with a 95% reduction for UspA1 50-491  and a 63% reduction for UspA1 299-452  ( FIG. 7 ). When fibronectin was pre-incubated with the truncated UspA2 101 318 , an inhibition of 50% was obtained. 
     Thus, the fibronectin binding domains of UspA1 and A2 block the interactions between fibronectin and  M. catarrhalis.    
     UspA1 299-452  and UspA2 165-318  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 ( FIG. 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 UspA1 299-452  and UspA2 165-318 . Thereafter,  M. catarrhalis  RH4 was added and bacterial adhesion analyzed. The relative adherence (measured by the number of colony forming units) after pre-incubation with 0.4 μmoles per 200 μl of UspA1 299 452 , UspA2 165-318 , 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 UspA1 433-580  and UspA2 30-177  did not inhibit the interactions between  M. catarrhalis  and the Chang epithelial cells ( FIG. 8B ). Thus, fibronectin on Chang epithelial cells may function as a receptor for  M. catarrhalis  and the amino acid residues 299-452 of UspA1 and 165-318 of UspA2 contain the ligand responsible for the interactions. 
     Interaction between  M. catarrhalis  and Laminin 
       M. catarrhalis  Binds Laminin through UspA1 and A2 
     Two clinical  M. catarrhalis  isolates (BBH18 and RH4) and their specific mutants lacking UspA1, UspA2 or both proteins were analyzed by a whole-cell ELISA.  M. catarrhalis  RH4 strongly bound to immobilized laminin.( FIG. 9A ). In contrast,  M. catarrhalis  RH4 uspA1 mutant (RH4ΔuspA1) showed a laminin binding of 89.9% of the wild type.  M. catarrhalis  RH4 uspA2 mutant (RH4ΔuspA2) and the double mutant RH4ΔuspA1/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 UspA1/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 UspA1, explaining the minimal difference in binding between the wild type and RH4ΔuspA1. Taken together, these results show that UspA1 and A2 bound laminin. 
     To further analyze the binding between UspA1/A2 and laminin, truncated UspA1 50-770  and UspA2 30-539  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 UspA1 50-770  and UspA2 3C-539  strongly bound soluble laminin and the binding was dose-dependent and saturable ( FIG. 9B ). 
     To define the laminin binding domains, recombinant UspA1 and A2 spanning the entire molecules were manufactured. Laminin was incubated with immobilized truncated UspA1 and A2 fragments and followed by quantification by ELISA. UspA1 50-491  bound to laminin almost as efficiently as UspA1 50-770  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, UspA2 3C-351 , was able to retain 44.7% binding capacity as compared to the full length protein. The shorter protein UspA2 3C-177  showed a 43.7% binding capacity. ( FIG. 10B ). These results show that the binding domains include residues found within the N-terminals of both UspA1 and UspA2. 
     Interaction between  M. catarrhalis  and C3 and C3met 
       M. catarrhalis  Outer Membrane Proteins UspA1 and UspA2 Inhibit both the Classical and the Alternative Pathway of the Complement Cascade 
     UspA2 surface expression is crucial for  M. catarrhalis  survival in normal human serum (NHS) [1, 58], i.e., moraxella UspA2 deficient mutants are rapidly killed when exposed to NHS. We have recently shown that both UspA1 and A2 bind C4BP and thus might inhibit the classical pathway of complement activation [58]. To further shed light on  M. catarrhalis  interactions with the complement system, survival of UspA1/A2 double mutants was studied in serum treated with either EGTA with addition of MgCl 2  (Mg-EGTA) or EDTA. Mg-EGTA inhibits the classical and lectin pathways and thus allows separate analysis of the alternative pathway. In contrast, EDTA inhibits all complement pathways by absorbing divalent cations (Mg 2+  and Ca 2+ ). The  M. catarrhalis  RH4 wild type survived after 30 min of incubation, whereas RH4ΔuspA1/A2 double mutant was killed by intact NHS after 10 min ( FIG. 12 ). When the classical pathway was inhibited (NHS+Mg-EGTA), the RH4ΔuspA1/A2 mutant survived for a significantly longer period of time as compared to NHS without any chelators, but not as long as the wild type bacterium. Furthermore, when both the classical and alternative pathways were blocked with EDTA,  M. catarrhalis  RH4ΔuspA1/A2 survived. A similar pattern was obtained with the  M. catarrhalis  BBH18 isolate and the corresponding BBH18 ΔuspA1/A2 mutants (not shown). In parallel, experiments with C1q and factor D/properdin deficient sera demonstrated that both the classical and the alternative pathways were inhibited by  M. catarrhalis  (not shown). Thus,  M. catarrhalis , a pathogen that frequently colonizes the human respiratory tract, does not only counteract the classical pathway but also the alternative pathway of the complement system by the outer membrane proteins UspA1 and A2. 
       M. catarrhalis  Absorbs C3 from EDTA-Inactivated Serum 
     C3b covalently binds to the surface of a microbe and hence induces the alternative pathway ( FIG. 11B ). To analyze whether  M. catarrhalis  can interact with C3, our RH4 wild type strain was incubated with NHS or NHS treated with EDTA. Binding or deposition (via covalent link) of C3/C3b at the bacterial surface of  M. catarrhalis  RH4 was detected by flow cytometry analysis with a polyclonal antibody (pAb) directed against C3d recognizing both C3 and C3b. Incubation of bacteria with NHS containing intact complement led to deposition of C3 ( FIG. 13 ). Interestingly, when the complement cascade was inactivated in the presence of EDTA, the  M. catarrhalis  RH4 still bound C3 ( FIG. 13A ).  Streptococcus pneumoniae  that was included for comparison did not absorb C3 from the EDTA-treated serum ( FIG. 13B ). In contrast to pneumococci,  M. catarrhalis  thus bound C3 irrespectively of complement activation. The internal thioester of C3 is spontaneously hydrolysed in fluid phase to C3(H 2 O). Thus, intact C3 or C3 (H 2 O) was the most likely forms of C3 interacting with  M. catarrhalis . Since  M. catarrhalis  also binds C4BP [58], we wanted to exclude that C4BP was involved in the C3 binding and for that purpose we used C4BP depleted serum.  M. catarrhalis  absorbed C3 from the C4BP depleted serum to the same extent as to NHS (not shown). 
     Binding of C3met to  M. catarrhalis  is Dose-Dependent and Non-Covalent 
     Our experiments implied that C3 bound to the surface of  M. catarrhalis  irrespectively of complement activation. Therefore, we analyzed whether converted C3, which is non-functional, could bind to the bacteria. Native C3 was purified from human serum and treated with methylamine, which converts C3 to a C3met molecule equivalent to C3b without the capacity to covalently bind to microbes ( FIG. 11C ). Flow cytometry analysis revealed that the  M. catarrhalis  RH4 wild type strain efficiently bound C3met in a dose-dependent and saturable manner ( FIGS. 14A  and B). This interaction was not mediated by the C3a part of the C3 molecule since C3b and C3(H 2 O) also bound  M. catarrhalis  (not shown). The binding between  M. catarrhalis  RH4 and C3met was based to a large extent on ionic interactions as increasing concentrations of NaCl inhibited the interaction ( FIG. 14C ). Similar results were obtained with the  M. catarrhalis  BBH18 wild type strain (not shown). 
     To determine whether the binding of C3 is a general feature of all  M. catarrhalis  strains, we selected a random series of clinical isolates (n=13) and analyzed their capacity to bind C3met. All  M. catarrhalis  strains bound C3met as revealed by a flow cytometry analysis with an anti-C3d pAb. The mfi values varied from 4 to 39. However,  S. pneumoniae  and  E. coli  that were included for comparison did not bind C3met. 
       M. catarrhalis  is a Unique C3 and C3met binding Bacterium 
     To extend our analysis of bacterial C3 absorption from NHS, related moraxella subspecies (n=13) as well as common human pathogens (n=13) were incubated in the presence of NHS-EDTA. Interestingly, among all the bacterial species tested,  M. catarrhalis  was the only bacterium binding C3 in complement-inactivated serum (Table 9). All related moraxella strains as well as the other human pathogens were also analyzed for binding of C3met. In parallel with the C3 binding,  M. catarrhalis  was the only species that bound C3met. Taken together,  M. catarrhalis  has a unique feature to strongly bind C3 and C3met in a non-covalent manner. 
       M. catarrhalis  Binds C3met via the Outer Membrane Proteins UspA1 and UspA2 
     To determine the  M. catarrhalis  protein responsible for the C3 binding, we tested a series of bacterial mutants devoid of the outer membrane proteins MID, UspA1 and/or UspA2 [22, 58]. Interestingly, the binding of C3met was significantly correlated with Usp expression ( FIG. 15 ).  M. catarrhalis  RH4Δmid bound C3met to the same degree as the wild type counterpart ( FIG. 15A-B ). The RH4ΔuspA1 mutant showed only a slightly decreased binding, whereas the RH4ΔuspA2 was a weaker binder as compared to the wild type counterpart ( FIG. 15C-D ). In parallel, C3met binding to the double RH4ΔuspA1/A2 mutant was completely abolished ( FIG. 15E ). Furthermore, when the same experiments were performed using NHS-EDTA, the same pattern was seen ( FIG. 15F-J ). When normal human serum was used, all mutants showed similar amount of C3 on their surface since it was a mixture of covalent deposition and binding of C3 ( FIG. 15K-O ). Similar results were obtained with the  M. catarrhalis □BBH18 isolate and the corresponding BBH18 mutants. 
     To further analyze the interaction between C3 and UspA1/A2, UspA1 50-770  and UspA2 30-539  were produced in  E. coli  and purified. The recombinant proteins were dot blotted onto a nitrocellulose membrane followed by incubation with iodine-labelled C3met. Recombinant MID 962-1200 , which is derived from the  M. catarrhalis  outer membrane protein MID [59], was included as a negative control. A weak binding to UspA1 50-770  was detected, whereas [ 125 I]-C3met strongly bound to UspA2 30-539  ( FIG. 16A ). These findings were further strengthened using surface plasmon resonance (i.e., Biacore). UspA1 50-770  and UspA2 30-539  were immobilized on the surface of a CM5 chip using amino coupling and C3met was injected until saturation was reached. The K D  for the interaction between C3met and UspA2 30-539  or UspA1 50-770  was 3 and 14 μM, respectively. In conclusion, we found that UspA2 was the major C3met-binding protein of  M. catarrhalis , whereas UspA1 contributed to the binding to a lower degree. 
     A C3 Binding Domain is Located between Amino Acid Residues 200 and 458 of UspA2 
     To define the C3 binding domain of UspA2, recombinant proteins spanning the entire UspA2 30-539  molecule were manufactured. C3met was incubated with the immobilized full length UspA1 50-770 , UspA2 30-539  and a series of truncated UspA2 proteins. Thereafter, the interactions were quantified by ELISA. In agreement with the dot blot experiments ( FIG. 16A ), UspA1 50-770  bound C3met to a much lower extent compared to UspA2 30-539  in the ELISA ( FIG. 16B ). Among the truncated protein fragments, UspA2 165-318 , UspA2 200-539  and UspA2 302-458  efficiently bound C3met, suggesting that a binding domain was within the amino acid residues 200 and 458. 
     Recombinant UspA1/A2 Neutralizes C3 Activity 
     In order to in detail examine the role of UspA1/A2-dependent inhibition of the alternative pathway, a series of flow cytometry experiments was performed with bacteria incubated with 10% NHS or serum that had been preincubated with 100 nM recombinant UspA1 50-770  and UspA2 30-539 . Interestingly, a significantly decreased C3 deposition/binding at the surface of  M. catarrhalis  RH4ΔuspA1/A2 was observed when NHS was pretreated with UspA1 50-770  and UspA2 30-539  ( FIG. 17A ). When the classical pathway was shut down with Mg-EGTA, similar results were obtained ( FIG. 17B ). Thus, the recombinant proteins UspA1 50-770  and UspA2 30-539  absorbed C3 from NHS and inhibited deposition/binding of C3. 
     To determine whether absorption of C3 by recombinant UspA1 50-770  and UspA2 30-539  increased bacterial survival, the double mutant  M. catarrhalis  RH4ΔuspA1/A2 was incubated with serum supplemented with UspA1 50-770  and UspA2 30-539  followed by determination of the number of surviving bacteria. Mg-EGTA was included in the reactions in order to inhibit the classical pathway. Interestingly, addition of recombinant UspA1 50-770  and UspA2 30-539  to NHS prevented killing of the UspA1/A2 deficient  M. catarrhalis  ( FIG. 17C ). UspA2 30-539  was most efficient in inhibiting bacterial killing as compared to UspA1 50-770 . When both recombinant proteins were supplemented together, no additional inhibition of the alternative pathway was detected. Ten % NHS correspond to approximately 600 nM C3. To investigate whether more UspA1 molecules could neutralize the C3 activity, UspA1 50-770  and/or UspA2 30-539  up to 600 nM was added. However, higher concentrations of the recombinant proteins did not further increase the inhibition (not shown). 
     We also included an alternative pathway haemolytic assay consisting of rabbit erythrocytes and NHS in order to establish the role of UspA1 and A2 as inhibitors of the alternative pathway. NHS was preincubated with recombinant UspA1 5C-770 , UspA2 30-539 , or both proteins together followed by addition to the erythrocytes. After 1 h incubation, the amount of erythrocyte lysis was determined. Interestingly, a significantly decreased haemolysis was observed when NHS was preincubated with UspA1 50-770  or UspA2 30-539  as compared to untreated NHS ( FIG. 18 ). In parallel with the increased survival of bacteria in the presence of UspA2 30-539  or UspA1 50-770  ( FIG. 17C ), preincubation with UspA2 30-539  alone resulted in a more efficient inhibition of the alternative pathway as compared to when NHS was preincubated with UspA1 50-770 . In conclusion, recombinant UspA1 50-770  or UspA2 30-539  interfered with the activity of the alternative pathway due to their ability to capture C3. 
     In addition of being a key molecule in the complement cascade, deposited C3b and iC3b (inactivated C3b) target microbes for removal in the process of opsonophagocytosis. To investigate whether C3 or C3met that was non-covalently bound at the surface of  M. catarrhalis  could still function as an opsonin, a series of phagocytosis experiments was performed.  M. catarrhalis  was preincubated with C3met, NHS or NHS treated with EDTA followed by addition of polymorphonuclear leukocytes. Interestingly,  M. catarrhalis  was not engulfed in the presence of C3met, whereas NHS strongly promoted phagocytosis (data not shown). However, when NHS was pretreated with EDTA,  M. catarrhalis  was not phagocytosed by polymorphonuclear leukocytes. Thus, C3/C3met was inactive at the  M. catarrhalis  cell surface and did not function as an opsonin. 
     DISCUSSION 
     Interaction between  M. catarrhalis  and Fibronectin 
     UspA1 299-452  and UspA2 165-318  from the clinical  M. catarrhalis  strain Bc5 were the shortest fragments that still bound fibronectin. Interestingly, longer fragments encompassing the amino acid sequence found within UspA1 299-452  and UspA2 165-318  displayed a more efficient binding to fibronectin ( FIGS. 5A  and B). This may mean that these two regions represent partial binding domains or that the binding site is highly dependent on a specific molecular structure. UspA1 299-452  and UspA2 165-318  share a sequence of 31 identical amino acid residues including the 23 residues “NNINNIYELAQQQDQHSSDIKTL” (SEQ ID NO: 85) (NNINNIY (SEQ ID NO: 86) sequence). This sequence contains the epitope for the protective monoclonal antibody (mAb) 17C7 for which there is universal reactivity. [2, 50, 30] In a mouse model, passive immunization with mAb 17C7 provided protection and improved pulmonary clearance of  M. catarrhalis.  [30] It is hence most interesting that UspA1/A2 fibronectin binding domains contain these residues and argues for the importance of this region in the pathogenesis of  M. catarrhalis  respiratory tract infection. 
     The fibronectin binding  M. catarrhalis  BBH18 and RH4 used in our experiments also carry the 31 amino acid residues in their UspA1/A2 protein. Most  M. catarrhalis  have a part of this sequence (i.e., the NNINNIY (SEQ ID NO: 86) sequence). However, strains like the O35E which has the NNINNIY (SEQ ID NO: 86) sequence in their UspA2 gene do not express a fibronectin binding UspA2 protein. [49] A likely explanation would be that the variations in the flanking regions might affect the interaction with fibronectin. Also, the conserved NNINNIY (SEQ ID NO: 86) sequence itself can have minor single amino acid base changes. [28] It is thus likely that fibronectin binding would depend not just on UspA1/A2 expression, but also on the individual makeup of each UspA protein. Interestingly, an almost identical amino acid sequence can be found in the hybrid UspA2H protein with adhesive properties [ M. catarrhalis  TTA37 and O46E). [43] This give support to our findings that the 31 amino acid sequence is important in adhesion. 
     In our last set of experiments, we tested whether the adherence of  M. catarrhalis  to Chang conjunctival cells could be inhibited by the fibronectin binding fragments (UspA1 299-452  and UspA2 165-318 ) ( FIG. 8B ). Preincubation with UspA1 299-452 , UspA2 165-318  or an anti-fibronectin pAb resulted in decreased binding to Chang epithelial cells. These results confirm the importance of these binding domains in the interactions of UspA1/A2 with Chang epithelial cells and further suggest that fibronectin is an important receptor for UspA. In addition, it is known that FnBP facilitate the adherence of bacteria to undifferentiated and injured airways.[54, 69] Fibronectin expression by lung fibroblasts is also increased by cigarette smoke extract.[87] The role of  M. catarrhalis  UspA1/A2 binding to ECM fibronectin or epithelial cell-associated fibronectin is thus of great importance in patients with COPD and may explain the common occurrence of  M. catarrhalis  infection in this group of patients.[40] 
     In conclusion, we have shown that UspA1/A2 of  M. catarrhalis  BBH18, RH4 and Bc5 are crucial FnBP. Both recombinant UspA1 and A2 derived from Bc5 bind fibronectin with a binding domain sharing identical amino acid residues including the conserved NNINNIY (SEQ ID NO: 86) sequence. Furthermore, an interaction of  M. catarrhalis  UspA1/A2 with epithelial cells is via cell-associated fibronectin. The definition of these fibronectin binding domains is therefore an important step forward in the development of a vaccine against  M. catarrhalis.    
     Interaction Between  M. catarrhalis  and 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 to laminin and thus 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] 
     We recently showed that both UspA1 and A2 bind fibronectin.[78] The fibronectin binding domains were located within UspA1 299-452  and UspA2 165-318 . In this study, the N-terminal halves UspA1 50-491  and UspA2 30-351  (containing the fibronectin domains) also bound laminin. However, the smallest fragments that bound fibronectin, UspA1 299-452  and UspA2 165-318  did not bind laminin to any appreciable extent. In fact, fragments smaller than the N-terminal half of UspA1 (UspA1 50-491 ) losses all its laminin binding ability whereas with UspA2, only UspA2 30-170  bound laminin albeit at a lower level then the whole recombinant protein (UspA2 30-539 ). These findings suggest that perhaps different parts of the molecules might have different functional roles. 
     Comparing the smallest laminin binding regions of UspA1 and A2, we find that there is, however, little similarity by way of amino acid homology between UspA2 30 170  and UspA1 50 491  (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] We postulate that a tertiary structure is likely responsible for the interactions with laminin in the head region in vivo. The localization of the binding domains at the N-terminal end would be logical as this would be most exposed and in contact with the human basement membrane in vivo. 
     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 UspA1/A2 bind both fibronectin and laminin, these proteins can be designated MSCRAMMS. Our results suggest that UspA1 and A2 are multifunctional adhesins with different domains interacting with different ligands in the respiratory tract. Similar broad-spectrum binding profiles have been reported for other bacterial proteins such as YadA of  Yersinia enterocolitica  for which UspA1 and A2 bear a structural relationship. [45, 70] YadA too binds both fibronectin and laminin. [32] 
     In summary we have shown that UspA1/A2 are crucial to  M. catarrhalis  interaction with the basement membrane glycoprotein laminin and this will play an important role in the pathogenesis of infections in patients with COPD. [74] 
     Interaction Between  M. catarrhalis  and C3 and C3met 
     Complement resistance is one of the most important bacterial virulence factors.[66] The majority (89%) of  M. catarrhalis  isolates from patients with lower respiratory tract infections are resistant to complement-mediated killing.[34]  M. catarrhalis  UspA1 and A2 are crucial for bacterial survival in human serum in vivo [1, 15], and we have shown that these two outer membrane proteins bind to the complement fluid phase regulator of the classical pathway, C4BP.[58] In the present study, we demonstrate that  M. catarrhalis  can inhibit the alternative pathway by non-covalently binding of C3 ( FIGS. 17 and 18 ). The binding of C3 most likely also inhibits the classical pathway. This could, however, not be analysed in detail since  M. catarrhalis  also binds C4BP. Interestingly, the  M. catarrhalis -dependent C3-binding is unique as several related moraxella subspecies as well as common human pathogenic bacteria do not bind C3 (Table 9). The interactions with C3 and methylamine-treated C3 are mediated mainly by UspA2, whereas UspA1 has a minor role ( FIGS. 15 and 16 ). The C3-binding region of UspA2 was localized between the amino acid residues 200 to 458. This region contains a stretch of 140 amino acid residues that is 93% identical to a region in UspA1.[2] However, despite this sequence similarity, UspA1 binds C3 to a much lower extent. This might be due to a specific difference in conformation between the proteins. The discrepancy in the C3 binding of UspA1 and UspA2 stands in contrast to the UspA1/A2 interaction with C4BP.[58] 
       M. catarrhalis  is equally resistant to both the classical and alternative pathways ( FIG. 12B ). The bacterium binds C4BP that inhibits the classical pathway [58] and in this paper we demonstrate an interaction with the alternative pathway through binding of C3. To determine which of these mechanisms that is of most importance for the  M. catarrhalis  serum resistance in various in vivo situations is difficult. For example, the importance of the classical pathway will strongly depend on history of infections with  M. catarrhalis  and ability to generate complement-activating antibodies. However, every mechanism providing protection from the complement is certainly beneficial for a pathogen. Since C3 is a key molecule in the complement system, the binding of C3 most likely results in regulation of all three activation pathways and may contribute the most to serum resistance. 
     The importance of the complement system as a primary defence mechanism is mirrored by the fact that microbes have developed various strategies to interfere with and/or neutralize components of the complement system.[42, 35, 88] In addition to  M. catarrhalis, S. pyogenes, Bordetella pertussis, E. coli K 1 , Candida albicans , and  N. gonorrhoeae  express specific surface molecules that bind C4BP and as a consequence protect the bacteria against the classical complement pathway.[8, 9, 52, 58, 64, 65, 80] In addition to inhibition of the classical pathway, several bacteria (e.g.,  C. albicans, N. meningitides, S. pyogenes , and  S. pneumoniae ; for reviews see [68, 89] bind factor H and factor H-like molecule and hence are partially protected against the alternative complement pathway. 
     UspA1 and A2 absorb C3 from serum and hereby most likely inhibit the complement activation. Similarly, the Pneumococcal Surface Protein A (PspA) appears to inhibit the alternative pathway both in vitro and in vivo. PspA is an important virulence factor for  S. pneumoniae . PspA-deficient pneumococcal strains are readily cleared from the blood, whereas the PspA-expressing strains survive.[82] Furthermore, in a murine model of bacteremia, PspA-deficient pneumococci have a significantly reduced virulence compared with pneumococci that express PspA.[11] It has been demonstrated that more C3b is deposited on PspA-negative pneumococci than on PspA-positive.[67, 82] Thus, expression of PspA reduces the complement-mediated clearance and phagocytosis of  S. pneumoniae  by limiting opsonization by C3b.[12, 67] PspA-deficient pneumococci that are not virulent in normal mice become virulent in C3-deficient and factor B-deficient mice.[82] 
     To our knowledge, there are only two examples of bacterial proteins that non-covalently bind C3 and thereby interfere with complement function. The first one is the extracellular fibrinogen-binding protein (Efb) of  Staphylococcus aureus , which was found to bind C3b.[44] Efb inhibits both the classical and alternative pathways independently of the thioester conformation, i.e., the binding to C3b is non-covalent. The second example is the pneumococcal choline-binding protein (CbpA), which has been shown to bind methylamine-treated C3, suggesting a non-covalent interaction that is not dependent on complement activation.[16] CbpA is a component of the pneumococcal cell wall, but may only bind C3 when the CbpA is secreted. In order to test this hypothesis, which is not firmly established in the literature, we analyzed eleven different pneumococcal isolates for C3 binding (methylamine-treated C3 or NHS-EDTA) by flow cytometry ( FIG. 12B  and Table 9). No bound C3 could be detected on the surface of  S. pneumoniae . When lysates of  S. pneumoniae  and culture supernatants were analyzed on Western blots using methylamine-treated C3 followed by an anti-human C3 pAb, we confirmed the results by Cheng and collaborators [16] (not shown). In the light of Efb and CbpA, which both are C3-binding proteins secreted by two Gram-positive bacteria, the Gram-negative  M. catarrhalis  is a unique species with membrane anchored proteins that bind C3 and inhibit the alternative pathway at the surface of the bacterium. 
     The yeast  Candida albicans  has been shown to bind C3b, iC3b and C3d. However, C3b is bound at a considerably lower affinity than iC3b and C3d.[29] We found a large difference between C3 binding to  M. catarrhalis  and  C. albicans  (not shown); despite that candida bound C3met (56% positive cells), the mean fluorescence intensity (mfi) was only &lt;2.0 as compared to mfi 36.9 for  M. catarrhalis . Furthermore, no detectable binding was seen when  C. albicans  was incubated with EDTA-treated serum. Two C3d-binding proteins have been isolated from  C. albicans  and the most characterized protein is a 60 kDa mannoprotein that initially was recognized by an antibody directed against human complement receptor 2 (CD21).[13] However,  M. catarrhalis  UspA1 and A2 were not recognized by a polyclonal antibody directed against CD21 (not shown). In parallel with staphylococci and pneumococci [52, 64], a secreted C3d-binding protein from  C. albicans  also exists.[72] Finally, a  C. albicans  iC3b receptor has been isolated and is structurally similar to human CR3 (CD11b).[3] The mechanisms by which these receptors may participate in pathogenesis are not fully known. 
     The above examples of C3 binding pathogens are notably different from  M. catarrhalis  in that these species often are blood stream isolates.  M. catarrhalis  is mucosal pathogen with rare instances of bacteremic infections. Hence, the binding and inactivating C3 most likely occur at the mucosal surface. This is supported by the fact that there is strong ongoing complement activation and consequent inflammation in disease state such as acute otitis media.[57] The complement proteins are believed to be transported to the mucosal surface due to exudation of plasma.[26, 62] In middle-ear effusions (MEEs) from children for example, strongly elevated concentrations of C3 products can also be found.[51] In addition, complement factors in MEEs fluid have been shown to be important in the bactericidal activity against other mucosal agents such as non-typable  H. influenzae. [75]  M. catarrhalis  is a strict human pathogen. It does not cause diseases such as otitis media or pneumonia in animals. A mouse pulmonary clearance model and an otitis media model with chinchilla has been used at several occasions. However, neither otitis media nor pneumonia develops and bacteria are rapidly cleared.[19, 83] It is thus difficult to test the biological significance of bacterial C3 binding in vivo. Since UspA1 and A2 are multifunctional proteins [1, 15, 31, 43, 58, 78], it would be impossible to relate any differences in the clearance of  M. catarrhalis  to C3 binding. In particular the fact that UspA1 is an important adhesin of  M. catarrhalis  and binds both CEACAM1 and fibronectin [31, 78] would most likely affect the clearance. Nevertheless, due to the strong complement activation in disease states such as otitis media, moraxella-dependent binding of C3 may represent an important way of combating the mucosal defense. 
     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 [73]. In conclusion,  M. catarrhalis  has developed sophisticated ways of combating both the humoral and innate immune systems. The present data show that  M. catarrhalis  has a unique C3-binding capacity at the bacterial cell surface that cannot be found in other bacterial species. 
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