Molecular vaccine linking intercellular spreading protein to an antigen

Abstract

Superior molecular vaccines comprise nucleic acids, including naked DNA and replicon RNA, that encode a fusion polypeptide that includes an antigenic peptide or polypeptide against which an immune response is desired. Fused to the antigenic peptide is an intercellular spreading protein, in particular a herpes virus protein VP22 or a homologue or functional derivative thereof. Preferred spreading proteins are VP22 from HSV-1 and Marek's disease virus. The nucleic acid can encode any antigenic epitope of interest, preferably an epitope that is processed and presented by MHC class I proteins. Antigens of pathogenic organisms and cells such as tumor cells are preferred. Vaccines comprising HPV-16 E7 oncoprotein are exemplified. Also disclosed are methods of using the vaccines to induce heightened T cell mediated immunity, in particular by cytotoxic T lymphocytes, leading to protection from or treatment of a tumor.

1 . A nucleic acid molecule encoding a fusion polypeptide useful as a vaccine composition, which molecule comprises:
 (a) a first nucleic acid sequence encoding a first polypeptide that comprises at least one intercellular transport polypeptide;   (b) optionally, fused in frame with the first nucleic acid sequence, a linker nucleic acid sequence encoding a linker peptide; and   (c) a second nucleic acid sequence that is linked in frame to said first nucleic acid sequence or to said linker nucleic acid sequence and that encodes an antigenic polypeptide or peptide.   
     
     
         2 . (canceled) 
     
     
         3 . The nucleic acid molecule of  claim 1 , wherein the antigenic polypeptide comprises an epitope that binds to, and is presented on the cell surface by, an MHC class I protein. 
     
     
         4 . (canceled) 
     
     
         5 . The nucleic acid molecule of  claim 1 , wherein the transport polypeptide is a viral polypeptide or a homologue thereof. 
     
     
         6 - 8 . (canceled) 
     
     
         9 . The nucleic acid molecule of  claim 1 , wherein the transport polypeptide comprises SEQ ID NO: 26. 
     
     
         10 . The nucleic acid molecule of  claim 1  wherein the transport polypeptide comprises SEQ ID NO: 28. 
     
     
         11 - 22 . (canceled) 
     
     
         23 . An expression vector comprising the nucleic acid molecule of  claim 1  operatively linked to
 (a) a promoter; and   (b) optionally, additional regulatory sequences that regulate expression of said nucleic acid in a eukaryotic cell.   
     
     
         24 - 30 . (canceled) 
     
     
         31 . A cell which has been modified to comprise the expression vector of  claim 23 . 
     
     
         32 - 34 . (canceled) 
     
     
         35 . A particle comprising the expression vector of  claim 23 . 
     
     
         36 . The particle of  claim 35  which comprises a material is suitable for introduction into a cell or an animal by particle bombardment. 
     
     
         37 . (canceled) 
     
     
         38 . A fusion polypeptide comprising
 (a) a first domain comprising an intercellular transport polypeptide and   (b) a second domain comprising an antigenic peptide or polypeptide.   
     
     
         39 - 46 . (canceled) 
     
     
         47 . A pharmaceutical composition capable of inducing or enhancing an antigen-specific immune response, comprising:
 (a) pharmaceutically and immunologically acceptable excipient in combination with;   (b) a composition selected from the group consisting of:
 (i) a nucleic acid molecule encoding a fusion polypeptide useful as a vaccine composition, which molecule comprises; 
 (ii) an expression vector comprising the nucleic acid molecule of (i) operably linked to a promoter, and optionally, an additional regulatory sequence that regulates expression of the nucleic acid molecule in a eukaryotic cell; 
 (iii) a cell comprising the nucleic acid molecule of (i) or the expression vector of (ii); 
 (iv) a particle comprising the nucleic acid molecule of (i), the expression vector of (iii), or the cell of claim (iii); 
 (v) a fusion polypeptide comprising; and
 (a) a first domain comprising an intercellular transport polypeptide and 
 (b) a second domain comprising an antigenic peptide or polypeptide. 
 
 (vi) any combination of (i)-(v). 
   
     
     
         48 - 50 . (canceled) 
     
     
         51 . A method of inducing or enhancing an antigen specific immune response in cells or in a subject comprising administering to said cells or to said subject an effective amount of the pharmaceutical composition of  claim 47 , thereby inducing or enhancing said response. 
     
     
         52 . The method of  claim 51 , wherein the composition is administered ex vivo to said cells. 
     
     
         53 . The method of  claim 52  wherein said cells comprise APCs. 
     
     
         54 . The method of  claim 53 , wherein said APCs are dendritic cells. 
     
     
         55 . The method of  claim 53 , wherein the APCs are human APCs. 
     
     
         56 . The method of  claim 53 , wherein the APCs are isolated from a living subject. 
     
     
         57 . The method of  claim 52 , further comprising a step of administering the ex vivo-treated cells to a histocompatible subject. 
     
     
         58 . The method of  claim 51  wherein said cells are human cells and said subject is a human. 
     
     
         59 . The method of  claim 51  wherein said administering is by a intramuscular, intradermal, or subcutaneous route. 
     
     
         60 . The method of  claim 51  wherein the composition comprises said nucleic acid molecule, said expression vector said cell, said fusion polypeptide or said particle, and said administering is by biolistic injection. 
     
     
         61 . The method of  claim 51 , wherein the administering is intratumoral or peritumoral. 
     
     
         62 . The method of  claim 51 , wherein the inducing or enhancing or enhancing the antigen specific immune response comprises increasing the numbers or lytic activity of CD8+ CTLs specific for a selected antigen in the subject. 
     
     
         63 . The method of  claim 51 , wherein the inducing or enhancing the antigen specific immune response results in inhibiting growth or preventing re-growth of a tumor in the subject. 
     
     
         64 . The method of  claim 63 , wherein said administering is intratumoral or peritumoral. 
     
     
         65 . The method of  claim 63 , further comprising treating said subject with radiotherapy or chemotherapy.