Compositions and Methods for Increasing Immunogenicity of Glycoprotein Vaccines

Abstract

The present invention relates to the microbial immunogens engineered to bear α-gal epitope(s) for induction of potent humoral and cellular immune responses when administered to subjects having anti-Gal antibodies. In one embodiment, the present invention provides compositions and methods for propagating influenza virus in human, ape, Old World monkey or bird cells that have been engineered to express an α1,3galactosyltransferase (α 1,3GT) gene to produce virions bearing hemagglutinin molecules containing α-gal epitopes, to increase the immunogenicity of the influenza virus. In another embodiment, the present invention provides fusion proteins between influenza virus hemagglutinin and a microbial peptide or protein of interest, and enzymatic processing of this fusion protein to carry α-gal epitopes, to increase the immunogenicity of the microbial peptide or protein of interest.

1 . A method, comprising:
 a) providing;
 i) an influenza virus; and 
 ii) a host cell susceptible to infection by said influenza virus and comprising an expression vector comprising a nucleic acid encoding an α1,3galactosyltransferase (α1,3gal) in operable combination with a promoter; and 
   b) inoculating said host cell to produce an inoculated host cell, wherein said inoculated host cell produces an influenza virus bearing an α-gal epitope (Galα1-3Galβ1-4(3)GlcNAc-R).   
     
     
         2 . The method of  claim 1 , wherein said influenza virus is an influenza A virus or an influenza B virus. 
     
     
         3 . The method of  claim 1 , wherein said host cell is selected from the group consisting of a human cell, an ape cell, an Old World monkey cell and a bird cell. 
     
     
         4 . The method of  claim 3 , wherein said Old World monkey cell is a Vero cell. 
     
     
         5 . The method of  claim 1 , wherein said α1,3gal is an enzyme of a species selected from the group consisting of a mouse, a cow, a cat, a sheep, a rat, a pig and a New World monkey. 
     
     
         6 . The method of  claim 5 , wherein said New World monkey is a common marmoset. 
     
     
         7 . The method of  claim 1 , further comprising step c) inactivating said influenza virus bearing an α-gal epitope to produce an inactivated influenza virus bearing an α-gal epitope. 
     
     
         8 . The method of  claim 7 , further comprising step d) administering said inactivated influenza virus bearing an α-gal epitope to a subject having anti-Gal antibodies under conditions suitable for induction of an immune response by said subject. 
     
     
         9 . The method of  claim 8 , wherein said immune response comprises production of antibodies reactive with said influenza virus and T lymphocytes reactive with cells infected by said influenza virus. 
     
     
         10 . A composition comprising an isolated host cell susceptible to infection by an influenza virus and comprising an expression vector comprising a nucleic acid encoding an α1,3galactosyltransferase (α1,3gal) in operable combination with a promoter. 
     
     
         11 . The composition of  claim 10 , wherein said isolated host cell has little or no sialytransferase activity. 
     
     
         12 . The composition of  claim 11 , wherein said isolated host cell is an inoculated host cell that produces an influenza virus bearing an α-gal epitope (Galα1-3Galβ1-4(3)GlcNAc-R). 
     
     
         13 . The composition of  claim 12 , wherein said influenza virus is an influenza A virus or an influenza B virus. 
     
     
         14 . A method of producing an isolated fusion protein comprising an amino-terminal portion and a carboxy-terminal portion, the method comprising:
 a) providing recombinant host cells comprising an expression vector, wherein said expression vector comprises a nucleic acid encoding said fusion protein in operable combination with a promoter, and wherein said amino-terminal portion comprises influenza virus hemagglutinin (HA) and said carboxy-terminal portion comprises a protein antigen of interest;   b) culturing said recombinant hosts cells to produce said fusion protein; and   c) isolating said fusion protein.   
     
     
         15 . The method of  claim 14 , wherein said influenza virus is an influenza A virus or an influenza B virus. 
     
     
         16 . The method of  claim 14 , wherein said protein antigen of interest comprises an influenza virus nucleoprotein. 
     
     
         17 . The method of  claim 14 , wherein said protein antigen of interest comprises an influenza virus M2e oligopeptide. 
     
     
         18 . The method of  claim 14 , further comprising incubating said fusion protein in the presence of neuraminidase, α1,3galactosyltransferase (α1,3gal) and UDP-galactose to produce a fusion protein comprising α-gal epitopes. 
     
     
         19 . The method of  claim 14 , wherein said transgenic host cell further comprises an expression vector comprising a nucleic acid encoding influenza virus neuraminidase in operable combination with a promoter to effect removal of sialic acid from said fusion protein to produce a sialic acid deficient fusion protein. 
     
     
         20 . The method of  claim 19 , wherein said method further comprises incubating said sialic acid deficient fusion protein in the presence of α1,3galactosyltransferase (α1,3gal) and UDP-galactose to produce a fusion protein comprising α-gal epitopes. 
     
     
         21 . The method of  claim 19 , wherein said transgenic host cell further comprises an expression vector comprising a nucleic acid encoding an α1,3galactosyltransferase (α1,3gal) in operable combination with a promoter to produce a fusion protein comprising α-gal epitopes. 
     
     
         22 . The fusion protein produced by the method of  claim 14 .