Method and reagents for genetic immunization

Abstract

DNA vaccines which incorporate genetic sequences encoding sorting signals which direct an expressed antigen to a specific cellular organelle facilitate loading of the antigen onto a Class I or Class II MHC molecule for immune presentation. These vaccines are a nucleic acid construct of a genetic sequence encoding a protein or peptide antigen and a sorting signal which will direct expressed antigen to the ER or endosomal-lysosomal compartments within the cell. The resulting constructs can be used as naked DNA vaccines, packaged in liposomes, or coated onto colloidal gold particles. The construct might also be delivered in an expression vector which is expressed in cells of the organism being immunized.

1 . A nucleic acid construct for genetic immunization comprising 
 (a) an antigen-coding region encoding an antigenic protein or peptide; and    (b) a sorting region encoding a protein or peptide which acts as a sorting signal to direct intracellular transport of the protein or peptide to the endosomes or the endoplasmic reticulum of a cell.    
     
     
         2 . The construct of  claim 1 , further comprising a linker region disposed between the antigen-coding region and the sorting region.  
     
     
         3 . The construct according to  claim 1  or  2 , wherein the sorting region is derived from the human brown locus protein, gp75; human albino locus protein, tyrosinase; human silver locus protein, Pmel 17; or human pink eyed locus P-protein.  
     
     
         4 . The construct according to  claim 1  or  2 , wherein the sorting region encodes at least the peptide Glu Ala Asn Gln Pro Leu Leu Thr Asp (SEQ ID NO. 1).  
     
     
         5 . The construct according to  claim 1  or  2 , wherein the sorting region encodes at least the peptide Glu Glu Lys Gln Pro Leu Leu Met Asp (SEQ ID NO. 2).  
     
     
         6 . The construct according to  claim 1  or  2 , wherein the sorting region encodes at least the peptide Glu Asp Ser Pro Leu Leu (SEQ ID NO. 3).  
     
     
         7 . The construct according to  claim 1  or  2 , wherein the sorting region encodes at least the peptide Glu Asp Thr Pro Leu Leu (SEQ ID NO. 4).  
     
     
         8 . The construct according to  claim 1  or  2 , wherein the sorting region encodes at least the peptide sequence Pro Ser Arg Asp Arg Ser Arg His Asp Lys Ile His (SEQ ID NO. 5).  
     
     
         9 . The construct according to  claim 1  or  2 , wherein the sorting region is a mutant form in which a glycosylation site present in a corresponding wild type sorting region has been altered.  
     
     
         10 . The construct according to any of  claims 1  to  9 , further comprising a promoter region effective to permit expression of the construct in mammalian cells.  
     
     
         11 . The construct according to  claim 10 , wherein the promoter region is selected from among the SV40 promoter, the CMV promoter and the RSV promoter.  
     
     
         12 . A vaccine for genetic immunization comprising a nucleic acid construct according to any of  claims 1  to  11 .  
     
     
         13 . The vaccine according to  claim 12 , wherein the nucleic acid construct is packaged in a liposome.  
     
     
         14 . The vaccine according to  claim 12 , wherein the nucleic acid construct is coated on a colloidal gold particle.  
     
     
         15 . The vaccine according to  claim 12 , wherein the nucleic acid construct is incorporated into a viral expression vector.  
     
     
         16 . A method for inducing an immune response to an antigen in a mammal, comprising the step of administering to the mammal a nucleic acid construct or vaccine according to any of claims  1 - 15 .  
     
     
         17 . A method for preparing a vaccine for genetic immunization comprising the step preparing a nucleic acid construct according to any of  claims 1  to  11 .  
     
     
         18 . The method according to  claim 17 , further comprising the step of packaging the nucleic acid construct in a liposome carrier.  
     
     
         19 . The method according to  claim 17 , further comprising the step of coating the nucleic acid construct on a colloidal gold particle.  
     
     
         20 . The method according to  claim 17 , wherein the nucleic acid construct is incorporated into a viral expression vector.