High molecular weight major outer membrane protein of Moraxella

Abstract

An isolated and purified outer membrane protein of a Moraxella strain, particularly M. catarrhalis , having a molecular mass of about 200 kDa, is provided. The about 200 kDa outer membrane protein as well as nucleic acid molecules encoding the same are useful in diagnostic applications and immunogenic compositions, particularly for in vivo administration to a host to confer protection against disease caused by a bacterial pathogen that produces the about 200 kDa outer membrane protein or produces a protein capable of inducing antibodies in a host specifically reactive with the about 200 kDa outer membrane protein.

What we claim is:  
     
         1 . An isolated and purified outer membrane protein of a Moraxella strain having an apparent molecular mass of about 200 kDa, as determined by SDS-PAGE or a fragment or an analog thereof.  
     
     
         2 . The protein of  claim 1  wherein the Moraxella strain is  Moraxella catarrhalis.    
     
     
         3 . The protein of  claim 2  wherein the strain is  Moraxella catarrhalis  4223.  
     
     
         4 . The protein of  claim 1  containing the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10) for  Moraxella catarrhalis  strain 4223 or containing the corresponding amino acid sequence from other Moraxella strains.  
     
     
         5 . The protein of  claim 1  which is at least about 70 wt % pure.  
     
     
         6 . The protein of  claim 5  which is at least about 95 wt % pure.  
     
     
         7 . The protein of  claim 1  in the form of an aqueous solution thereof.  
     
     
         8 . The protein of  claim 1  recognizable by an antibody preparation specific for a peptide having the amino acid sequence of NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10).  
     
     
         9 . The protein of  claim 1  having substantially the amino acid composition as shown in Table III.  
     
     
         10 . A purified and isolated nucleic acid molecule encoding an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or a fragment or an analog of the outer membrane protein.  
     
     
         11 . The nucleic acid molecule of  claim 10 , wherein the strain of Moraxella is a strain of  Moraxella catarrhalis.    
     
     
         12 . The nucleic acid molecule of  claim 11 , wherein the strain is  Moraxella catarrhalis  4223.  
     
     
         13 . The nucleic acid molecule of  claim 10 , wherein the encoded protein contains the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10) for  Moraxella catarrhalis  strain 4223 or contains the corresponding amino acid sequence from other Moraxella strains.  
     
     
         14 . A purified and isolated nucleic acid molecule having a sequence selected from the group consisting of: 
 (a) a DNA sequence as set out in FIG. 6 (SEQ ID Nos: 1 and 2), or the complementary sequence thereto;    (b) a DNA sequence encoding a 200 kDa protein of a strain of Moraxella and containing the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10), or the complementary sequence thereto;    (c) a DNA sequence encoding a deduced amino acid sequence as set out in FIG. 6 (SEQ ID No: 3), or the complimentary sequence thereto; and    (d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c).    
     
     
         15 . The nucleic acid molecule of  claim 14 , wherein the nucleotide sequence defined in (d) has at least about 90% sequence identity with any one of the sequences defined in (a), (b) or (c).  
     
     
         16 . A vector adapted for transformation of a host comprising the nucleic acid molecule of  claim 10  or  14 .  
     
     
         17 . An expression vector adapted for transformation of a host comprising the nucleic acid molecule of  claim 10  or  14  and expression means operatively coupled to the nucleic acid molecule for expression by the host of said outer membrane protein of a strain of Moraxella or the fragment or the analog of the outer membrane protein.  
     
     
         18 . The expression vector of  claim 17 , wherein the expression means includes a nucleic acid portion encoding a leader sequence for secretion from the host of the outer membrane protein or the fragment or the analog of the outer membrane protein.  
     
     
         19 . The expression vector of  claim 17 , wherein the expression means includes a nucleic acid portion encoding a lipidation signal for expression from the host of a lipidated form of the outer membrane protein or the fragment or the analog of the outer membrane protein.  
     
     
         20 . A transformed host containing an expression vector as claimed in  claim 17 .  
     
     
         21 . A recombinant outer membrane protein or fragment or analog thereof producible by the transformed host of  claim 20 .  
     
     
         22 . A live vector for delivery of an outer membrane protein of a strain of Moraxella having a molecular weight of about 200 kDa or a fragment or analog thereof to a host, comprising a vector containing the nucleic acid molecule of  claim 10  or  14 .  
     
     
         23 . The live vector of  claim 21 , wherein the vector is selected from the group consisting of  E. coli , Salmonella, Mycobacteria, adenovirus, poxyirus, vaccinia and poliovirus.  
     
     
         24 . A peptide having no less than six amino acids and no more than 150 amino acids and containing an amino acid sequence corresponding to a portion only of an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or a fragment or of an analog of the outer membrane protein.  
     
     
         25 . The peptide of  claim 24 , wherein the Moraxella strain is a  Moraxella catarrhalls  strain.  
     
     
         26 . The peptide of  claim 25 , wherein the strain is  Moraxella catarrhalis  4223.  
     
     
         27 . The peptide of  claim 24  having the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile (SEQ ID No: 5) or NH 2 -Asn-Val-Lys-Ser-Val-lle-Asn-Lys-GlU-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQ ID No: 6) for the  Moraxella catarrhalis  4223 strain or the amino acid sequence for the corresponding peptide for other strains of Moraxella.  
     
     
         28 . An immunogenic composition, comprising at least one active component selected from the group consisting of: 
 (A) an isolated and purified outer membrane protein of a Moraxella strain having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or a fragment or an analog thereof;    (B) a purified and isolated nucleic acid molecule encoding an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE or a fragment or an analog thereof;    (C) a purified and isolated nucleic acid molecule having a sequence selected from the group consisting of: 
 (a) a DNA sequence set out in FIG. 6 (SEQ ID No: 1 or 2), or the complementary sequence thereto;  
 (b) a DNA sequence encoding an about 200 kDa protein of a strain of Moraxella and containing the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10), or the complementary sequence thereto;  
 (c) a DNA sequence encoding an amino acid sequence as set forth in FIG. 6 (SEQ ID No: 3), or the complimentary sequence thereto; and  
 (d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c);  
   (D) a recombinant outer membrane protein or fragment or analog thereof producible in a transformed host containing an expression vector comprising a nucleic acid molecule as defined in (B) or (C) and expression means operatively coupled to the nucleic acid molecule for expression by the host of the recombinant outer membrane protein or fragment or analog thereof;    (E) a live vector, comprising a vector containing a purified and isolated nucleic acid molecule encoding a protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or a fragment or analog thereof;    (F) a live vector, comprising a vector containing a purified and isolated nucleic acid molecule having a sequence selected from the group consisting of: 
 (a) a DNA sequence as set out in FIG. 6 (SEQ ID No: 1), or the complementary sequence thereto;  
 (b) a DNA sequence encoding an about 200 kDa protein of a strain of Moraxella and containing the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10), or the complementary sequence thereto;  
 (c) a DNA sequence encoding an amino acid sequence as set forth in FIG. 6 (SEQ ID No: 3), or the complimentary sequence thereto; and  
 (d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c); and  
   (G) a peptide having no less than six amino acids and no more than 150 amino acids and containing an amino acid sequence corresponding to a portion only of an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or of an analog of the outer membrane protein; and    a pharmaceutically acceptable carrier therefor, said at least one active component producing an immune response when administered to a host.    
     
     
         29 . The immunogenic composition of  claim 28  formulated as a vaccine for in vivo administration to a host to confer protection against disease caused by a strain of Moraxella.  
     
     
         30 . The immunogenic composition of  claim 29  wherein the strain is  Moraxella catarrhalis.    
     
     
         31 . The immunogenic composition of  claim 29  formulated as a microparticle, capsule, ISCOM, or liposome preparation.  
     
     
         32 . The immunogenic composition of  claim 29  in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces.  
     
     
         33 . The immunogenic composition of  claim 29  further comprising at least one other immunogenic or immunostimulating material.  
     
     
         34 . The immunogenic composition of  claim 33  wherein the at least one other immunostimulating material is at least one adjuvant.  
     
     
         35 . The immunogenic composition of  claim 34  wherein the at least one adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein.  
     
     
         36 . The immunogenic composition of  claim 35  wherein the host is a primate.  
     
     
         37 . The immunogenic composition of  claim 36  wherein the primate is a human.