Vaccines against influenza virus

Abstract

Disclosed are immunogenic conjugates having the general formula: M2e-Cys-S—CH 2 —C(O)—NH—CH 2 —CH2-C(O—)NH-Lys-Pr, were M2e is the influenza M2 ectodomain (M2e) peptide; Cys is a cysteine amino acid residue present in the M2e peptide; S the sulfur present in the cysteine amino acid residue; CH2-CO—NH—CH2-CH2-CO the linking group; NH the amine group present in a lysine residue of the carrier; Lys is a lysine amino acid residue and Pr the carrier protein. Also disclosed are isolated immunogens that include an immunogenic fragment of an influenza HA protein including the polybasic cleavage site, wherein the immunogenic fragment of the influenza HA protein has been modified to remove an N-terminal leader amino acid sequence and a C-terminal transmembrane domain. Also disclosed are methods producing an influenza vaccine specific for an identified influenza strain.

We claim: 
     
         1 . A method of treating and/or inhibiting an influenza infection in a subject, comprising:
 selecting a subject for treatment that has, or is at risk for developing, an influenza infection; and   administering to a subject a therapeutically effective amount of an immunogenic conjugate comprising an influenza M2 ectodomain (M2e) peptide covalently linked to a carrier by a thioether linkage between a lysine amino acid residue present in carrier and a cysteine amino acid residue introduced at the C-terminal end of the M2e peptide, thereby treating and/or inhibiting the influenza infection in a subject.   
     
     
         2 . A method of producing an influenza vaccine specific for an identified influenza strain, the method comprising,
 obtaining a nucleic acid sequence of the identified influenza strain encoding a hemagglutinin (HA) from the identified influenza strain;   producing a nucleic acid molecule encoding an immunogenic fragment of the HA, wherein the nucleic acid molecule produced encodes the polybasic cleavage site, and wherein the nucleic acid molecule produced does not encode a leader sequence at the N-terminal end of the HA or a transmembrane domain at the C-terminal end of immunogenic fragment of the HA;   expressing the immunogenic fragment of the HA in a bacterial expression system; and   purifying the expressed immunogenic fragment of the HA, thereby producing an influenza vaccine specific for an identified influenza strain.   
     
     
         3 . The method of  claim 2 , further comprising adsorbing the purified immunogenic fragment of the influenza HA protein onto alum. 
     
     
         4 . The method of  claim 2 , further comprising treating the immunogenic fragment of the influenza HA protein with a fixative. 
     
     
         5 . The method of  claim 4 , wherein the fixative comprises formalin. 
     
     
         6 . The method of  claim 2 , further comprising adding a pharmaceutically acceptable carrier to the purified immunogenic fragment of the influenza HA protein. 
     
     
         7 . The method of  claim 2 , wherein the bacterial expression system is an  E. coli  expression system. 
     
     
         8 . The method of  claim 7 , wherein the nucleic acid molecule encoding an immunogenic fragment of the HA is codon optimized for expression in  E. coli.    
     
     
         9 . The method of  claim 2 , wherein the purified immunogenic fragment of the influenza HA protein is not glycosylated. 
     
     
         10 . The method of  claim 2 , wherein the immunogenic fragment of an influenza HA protein comprises a six residue histidine tag linked by a peptide linker to the C-terminal end of the immunogenic fragment of the influenza HA protein. 
     
     
         11 . The method of  claim 10 , wherein the peptide linker comprises Gly-Gly-Gly. 
     
     
         12 . An isolated immunogen comprising an immunogenic fragment of an influenza HA protein including the polybasic cleavage site, wherein the immunogenic fragment of the influenza HA protein has been modified to remove an N-terminal leader amino acid sequence and a C-terminal transmembrane domain. 
     
     
         13 . The isolated immunogen of  claim 12 , wherein the immunogenic fragment of the influenza HA protein is not glycosylated. 
     
     
         14 . The isolated immunogen of  claim 12 , wherein the immunogenic fragment of an influenza HA protein comprises a six residue histidine tag linked by a peptide linker to the C-terminal end of the immunogenic fragment of the influenza HA protein. 
     
     
         15 . The isolated immunogen of  claim 14 , wherein the peptide linker comprises Gly-Gly-Gly. 
     
     
         16 . The isolated immunogen of  claim 12 , further comprising an adjuvant. 
     
     
         17 . The isolated immunogen of  claim 16 , wherein the adjuvant comprise alum, and the immunogenic fragment of the influenza HA protein is adsorbed onto the alum. 
     
     
         18 . The isolated immunogen of  claim 12 , wherein the immunogenic fragment of the influenza HA protein is treated with a fixative. 
     
     
         19 . The isolated immunogen of  claim 18 , wherein the fixative comprises formalin. 
     
     
         20 . An immunogenic composition comprising the isolated immunogen of  claim 12  and a pharmaceutically acceptable carrier. 
     
     
         21 . A method of eliciting an immune response against an influenza antigenic epitope in a subject, comprising administering to the subject the immunogen of  claim 12 , thereby eliciting an immune response in the subject. 
     
     
         22 . The method of  claim 21 , wherein the immune response is elicited against an influenza HA protein. 
     
     
         23 . A method of treating and/or inhibiting an influenza infection in a subject, comprising:
 selecting a subject for treatment that has, or is at risk for developing, an influenza infection;   administering to a subject a therapeutically effective amount of the immunogen of  claim 12 , thereby treating and/or inhibiting the influenza infection in a subject.   
     
     
         24 . The method of  claim 1 , wherein the carrier is comprises bovine serum albumin, recombinant  B. anthracis  protective antigen, recombinant  P. aeruginosa  exotoxin A, tetanus toxoid, recombinant diphtheria toxoid, pertussis toxoid,  C. perfringens  toxoid, keyhole limpet hemocyanin, horseshoe crab hemocyanin, edestin, mammalian serum albumins, mammalian immunoglobulins, or analogs or mimetics of and combinations of two or more thereof. 
     
     
         25 . The immunogenic conjugate of  claim 24 , wherein the carrier comprises recombinant diphtheria toxoid (rDT). 
     
     
         26 . The immunogenic conjugate of  claim 1 , wherein the M2e peptide comprises the amino acid sequence set forth as X 1 LLTEVETX 2 X 3 X 4 X 5 X 6 WX 7 CX 8 X 9 X 10 X 11 X 12 X 13 X 14 C (SEQ ID NO: 3), where X 1  can be serine or valine; X 2  can be proline, leucine or histidine; X 3  can be isoleucine or threonine; X 4  can be arginine or lysine; X 5  can be asparigine or serine; X 6  can be glutamic acid or glycine; X 7  can be glycine or glutamic acid; X 8  can be arginine or lysine; X 9  can be cysteine or tyrosine; X 10  can be glutamine or serine; X 11  can be aspartic acid or glycine; X 12  can be serine or leucine; X 13  can be serine or arginine; and X 14  can be aspartic acid or glutamic acid. 
     
     
         27 . The immunogenic conjugate of  claim 26 , wherein the M2e peptide consists of the amino acid sequence set forth as X 1 LLTEVETX 2 X 3 X 4 X 5 X 6 WX 7 CX 8 X 9 X 10 X 11 X 12 X 13 X 14 C (SEQ ID NO: 3), where X 1  can be serine or valine; X 2  can be proline, leucine or histidine; X 3  can be isoleucine or threonine; X 4  can be arginine or lysine; X 5  can be asparigine or serine; X 6  can be glutamic acid or glycine; X 7  can be glycine or glutamic acid; X 8  can be arginine or lysine; X 9  can be cysteine or tyrosine; X 10  can be glutamine or serine; X 11  can be aspartic acid or glycine; X 12  can be serine or leucine; X 13  can be serine or arginine; and X 14  can be aspartic acid or glutamic acid. 
     
     
         28 . The immunogenic conjugate of  claim 26 , wherein the M2e peptide comprises the amino acid sequence set forth as SLLTEVETPTRNEWECRCSDSSDC (SEQ ID NO: 4). 
     
     
         29 . The immunogenic conjugate of  claim 1 , wherein the average ratio of M2e peptide molecules to carrier protein molecules is between about 1:1 and 15:1. 
     
     
         30 . The immunogenic conjugate of  claim 1 , wherein the average ratio of M2e peptide molecules to carrier protein molecules is between about 4:1 and 10:1. 
     
     
         31 . The immunogenic conjugate of  claim 1 , wherein the average ratio of M2e peptide molecules to carrier protein molecules is between about 4:1 and 7:1.