Universal influenza vaccine based on heterologous multiple m2e proteins

Abstract

Disclosed are universal influenza A vaccines capable of providing broader cross-protection. The vaccine contains a fusion protein comprising tandem repeats of heterologous M2e epitope sequences that have been molecularly and genetically designed to provide broad cross-protection. The fusion protein may be incorporated into virus-like particles (VLPs) or a replicating live attenuated influenza virus vaccine, and administered alone or in combination with other influenza vaccines.

1 - 2 . (canceled) 
     
     
         3 . The isolated polynucleotide of  claim 36 , wherein the human M2e domain comprises the amino acid sequence SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1. 
     
     
         4 . The isolated polynucleotide of  claim 36 , wherein the swine M2e domain comprises the amino acid sequence SEQ ID NO:2 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:2. 
     
     
         5 . The isolated polynucleotide of  claim 36 , wherein the avian M2e domains are from H5, H7, or H9 influenza A subtypes. 
     
     
         6 . The isolated polynucleotide of  claim 36 , wherein the avian M2e domain comprises the amino acid sequence SEQ ID NO:3 or SEQ ID NO:4 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3 or SEQ ID NO:4. 
     
     
         7 . The isolated polynucleotide of  claim 36 , wherein at least one M2e domain comprises an avian M2e domain comprising the amino acid sequence SEQ ID NO:3 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3, and wherein at least one M2e domain comprises an avian M2e domain comprising the amino acid sequence SEQ ID NO:4 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:4. 
     
     
         8 . The isolated polynucleotide of  claim 36 , wherein the fusion protein further comprises a signal peptide at the N-terminus. 
     
     
         9 . The isolated polynucleotide of  claim 8 , wherein the signal peptide comprises mellitin signal peptide. 
     
     
         10 . The isolated polynucleotide of  claim 9 , wherein the signal peptide comprises the amino acid sequence SEQ ID NO: 12 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:12. 
     
     
         11 . The isolated polynucleotide of  claim 36 , wherein the fusion protein further comprises an oligomer stabilization domain. 
     
     
         12 . The isolated polynucleotide of  claim 11 , wherein the oligomer stabilization domain comprises a leucine zipper tetramerization motif. 
     
     
         13 . The isolated polynucleotide of  claim 12 , wherein the oligomer stabilization domain comprises GCN4. 
     
     
         14 . The isolated polynucleotide of  claim 13 , wherein the oligomer stabilization domain comprises the amino acid sequence SEQ ID NO:13 or a an amino acid sequence having at least 90% sequence identity to SEQ ID NO:13. 
     
     
         15 . The isolated polynucleotide of  claim 36 , wherein the fusion protein further comprises a membrane anchor. 
     
     
         16 . The isolated polynucleotide of  claim 15 , wherein the membrane anchor is a transmembrane domain and optionally a cytoplasmic domain of a viral envelope protein. 
     
     
         17 . The isolated polynucleotide of  claim 16 , wherein viral envelope protein is an influenza A hemagglutinin (HA). 
     
     
         18 . The isolated polynucleotide of  claim 16 , wherein the transmembrane domain comprises the amino acid sequence SEQ ID NO:14 or SEQ ID NO:15 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:14 or SEQ ID NO:15. 
     
     
         19 . The isolated polynucleotide of  claim 36 , wherein the fusion protein comprises at least five heterologous M2e domains. 
     
     
         20 . The isolated polynucleotide of  claim 19 , wherein the fusion protein comprises at least ten heterologous M2e domains. 
     
     
         21 . The isolated polynucleotide of  claim 36 , wherein the fusion protein comprises an amino acid sequence having a formula selected from the group consisting of:
   X 1 -([hM2e] n -[sM2e] n -[aM2e] n ) n -X 2 -X 3 ,     X 1 -([hM2e] n -[aM2e] n -[sM2e] n ) n -X 2 -X 3 ,     X 1 -([sM2e] n -[hM2e] n -[aM2e] n ) n -X 2 -X 3 ,     X 1 -([sM2e] n -[aM2e] n -[hM2e] n ) n -X 2 -X 3 ,     X 1 -([aM2e] n -[sM2e] n -[hM2e] n ) n -X 2 -X 3 , and     X 1 -([aM2e] n -[hM2e] n -[sM2e] n ) n -X 2 -X 3 ;   wherein “X 1 ” consists of nothing or a signal peptide,   wherein “hM2e” consists of a human M2e domain,   wherein “sM2e” consists of a swine M2e domain,   wherein “sM2e” consists of an avian M2e domain,   wherein “X 2 ” consists of nothing or an oligomer stabilization domain,   wherein “X 3 ” consists of nothing or a membrane anchor domain,   wherein each “n” is independently an integer from one to five, and   wherein“-” consists of a peptide linker or a peptide bond.   
     
     
         22 . The isolated polynucleotide of  claim 21 , wherein the fusion protein comprises an amino acid sequence having the following formula:
   X 1 -(hM2e-hM2e-sM2e-aM2e-aM2e) n -X 2 -X 3 .   
     
     
         23 . The isolated polynucleotide of  claim 21  or  22 , wherein the signal peptide comprises melittin. 
     
     
         24 . The isolated polynucleotide of  claim 21 , wherein the oligomer stabilization domain comprises GCN4. 
     
     
         25 . The isolated polynucleotide of  claim 21 , wherein the membrane anchor domain comprises the transmembrane-cytoplasmic domain from an influenza A hemaglutinin. 
     
     
         26 . The isolated polynucleotide of  claim 22 , wherein “n” is 2. 
     
     
         27 . The isolated polynucleotide of  claim 36 , wherein the fusion protein comprises the amino acid sequence SEQ ID NO:16, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:16. 
     
     
         28 . The isolated polynucleotide of  claim 36 , wherein the VLP comprises matrix protein 1 (M1). 
     
     
         29 . The isolated polynucleotide of  claim 28 , wherein the vaccine is produced by co infecting insect cells with one or more recombinant baculoviruses expressing the M1 proteins and the fusion proteins, culturing the insect cells under physiological conditions, and purifying the VLPs from insect cell culture supernatants. 
     
     
         30 . The isolated polynucleotide of  claim 28 , wherein influenza virus hemagglutinin (HA) and neuraminidase (NA) are not immobilized on the surface of the VLP. 
     
     
         31 . The isolated polynucleotide of  claim 36 , further comprising an influenza virus-like particle (VLP) vaccine, a whole inactivated virus, split viral vaccine, or live attenuated influenza vaccine. 
     
     
         32 . The isolated polynucleotide of  claim 36 , formulated for delivery via intranasal, intramuscular, subcutaneous, transdermal or sublingual administration. 
     
     
         33 . The isolated polynucleotide of  claim 36 , further comprising an adjuvant. 
     
     
         34 . The isolated polynucleotide of  claim 33 , wherein the adjuvant is selected from the group consisting of ASO4 (alum plus monophosphoryllipid A), bacterial cell wall components, MF59 (mineral oil based adjuvant), and a molecular adjuvant incorporated VLP in a membrane-anchored form. 
     
     
         35 . The isolated polynucleotide adjuvant of  claim 34 , wherein the molecular adjuvant is GM-CSF (granulocyte macrophage colony stimulating factor) or bacterial flagellin. 
     
     
         36 . An isolated polynucleotide comprising a nucleic acid sequence encoding a fusion protein,
 wherein the fusion protein comprises three or more heterologous influenza virus matrix protein 2 extracellular (M2e) domains, and   wherein the fusion protein comprises one or more M2e domains from a human influenza A subtype, one or more M2e domains from a swine influenza A subtype, and one or more M2e domains from an avian influenza A subtype.   
     
     
         37 . The isolated polynucleotide of  claim 36 , wherein the nucleic acid sequence encoding the fusion protein is operably linked to an expression control sequence. 
     
     
         38 . A cell comprising the isolated polynucleotide of  claim 37 . 
     
     
         39 . The cell of  claim 38 , wherein the cell is a bacterium, insect cell, or yeast cell. 
     
     
         40 - 45 . (canceled)