DNA expression systems based on alphaviruses

Abstract

The disclosure describes recombinant alphavirus RNA molecules and expression of heterologous proteins therefrom in animal cells. Recombinant alphaviruses of the present invention, when made to express an antigenic protein, can be administered as vaccines.

1 . An RNA molecule derived from an alphavirus RNA genome and capable of efficient infection of animal host cells, which RNA molecule comprises the complete alphavirus RNA genome regions, which are essential to replication of the said alpha-virus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into a region of the RNA molecule which is non-essential to replication thereof.  
     
     
         2 . The RNA of  claim 1 , wherein the said alphavirus is Semliki Forest virus (SFV).  
     
     
         3 . The RNA of  claim 1  or  2 , wherein the exogenous RNA sequence encodes a protein, a polypeptide or a peptide sequence defining an exogenous antigenic epitope or determinant.  
     
     
         4 . The RNA of  claim 3  wherein the exogenous RNA sequence encodes an epitope sequence of a structural protein of an immunodeficiency virus inclusive of the human immunodeficiency virus (HIV) types.  
     
     
         5 . The RNA of any preceding claim, wherein the alphavirus derived RNA molecule regions comprise a 5′ terminal portion, the coding region(s) for non structural proteins required for RNA replication, the subgenome promoter region and a 3′ terminal portion of said viral RNA.  
     
     
         6 . The RNA of  claim 2 ,  3  or  5 , wherein the exogenous RNA sequence encodes a foreign polypeptide or protein and is integrated into the SFV subgenomic 26S RNA substituting deleted parts thereof.  
     
     
         7 . The RNA of  claim 2 ,  3 ,  4  or  5 , wherein the exogenous RNA sequence encodes a foreign viral epitopic peptide sequence and is located in a region of the RNA coding for structural alphavirus proteins enabling the exogenous RNA to be expressed as said viral epitope as part of the matured virus particle.  
     
     
         8 . The RNA of  claim 2 ,  3 ,  4  or  5 , wherein the exogenous RNA sequence encodes a foreign viral epitopic peptide sequence inserted into the p62 spike precursor subunit encoding region of the SFV genome.  
     
     
         9 . An RNA expression vector comprising the RNA of any preceding claim packaged into infectious particles comprising the RNA within the alphavirus nucleocapsid and surrounded by membrane with alphavirus spike proteins.  
     
     
         10 . The vector of  claim 9 , wherein the RNA has a total size corresponding to the wild type alphavirus RNA genome or deviating therefrom to an extent compatible with package of the RNA into the infectious particles.  
     
     
         11 . DNA transcription vector comprising a cDNA having one strand complementary to the RNA of any of claims  1  to 8.  
     
     
         12 . A DNA expression vector comprising a full-length or partial cDNA complementary to alphavirus RNA or parts thereof and located immediately downstream of the SP6 RNA polymerase promoter and having a 5′ATGG or 5′GATGG or any other 5′ terminus and a TTTCCA 69 ACTAGT or any other 3′ terminus.  
     
     
         13 . The vector of  claim 12  having portions of the viral cDNA deleted, the deletions comprising the complete or part of the region(s) encoding the virus structural proteins, and further comprising an integrated polylinker region, which may correspond to BamHI-SmaI-XmaI, inserted at a location which enables an exogenous DNA fragment encoding a foreign polypeptide or protein to be inserted into the vector cDNA for subsequent expression in an animal host cell.  
     
     
         14 . The vector of  claim 12  or  13  wherein the alphavirus is SFV.  
     
     
         15 . The vector of  claim 12  or  14  comprising full-length cDNA and further comprising an exogenous DNA fragment encoding a foreign epitopic peptide sequence or antigenic determinant inserted into a region of the viral structural proteins.  
     
     
         16 . The vector of  claim 15  wherein the exogenous DNA fragment is inserted into the p62 spike precursor subunit encoding region of the SFV cDNA.  
     
     
         17 . The vector of any preceding claim comprising an SFV derived cDNA which carries a conditionally lethal SFV mutation in the region encoding the p62 cleavage site, a cellularly uncleavable but extracellularly cleavable form of p62 being expressed.  
     
     
         18 . The vector of  claim 13  comprising SFV-derived cDNA, the vector being pSFV1, pSFV2 or pSFV3 having a structure as shown in FIG. 8.  
     
     
         19 . An RNA transcript derived from transcription of the DNA-vector of any of claims  12 - 18  carrying an exogenous DNA fragment.  
     
     
         20 . A method to produce the vector of  claim 9  or  10  wherein the alphavirus derived RNA lacks part of or the complete region(s) encoding the structural viral proteins, the method comprising cotransfection of animal host cells with the RNA transcript of  claim 19 , wherein the alphavirus RNA lacks part(s) of or the complete region(s) encoding the viral structural proteins, with helper RNA transcribed in vitro from a helper DNA vector and culturing the host cells.  
     
     
         21 . The method of  claim 20  wherein the cotransfection is produced by electroporation of the host cells.  
     
     
         22 . Helper vector for use in the method according to  claim 20  or  21 , said vector being comprised of the DNA vector of  claim 12  wherein the regions encoding non structural virus proteins are almost completely deleted, including sequences encoding RNA signals for packaging of RNA into nucleocapsid particles, but the 5′ and 31 signals needed for RNA replication and the region encoding the promoter for the structural sub-genome are in addition to those encoding the structural region preserved.  
     
     
         23 . Helper vector of  claim 22  wherein the cDNA has its origin from SFV and the deletion extends from the AccI (308) to the AccI (6399) restriction endonuclease site of the full-length cDNA vector of  claim 12 .  
     
     
         24 . Helper vector of claims  22  and  23  where the structural region contains the mutation described in  claim 17  or another conditionally lethal mutation.  
     
     
         25 . The method of  claim 20  wherein cells transformed to produce helper RNA according to claims  20 ,  22  or  23  are transfected with RNA transcript of  claim 19 .  
     
     
         26 . A host cell of animal origin transformed with the RNA of any of claims  1 - 8 , the DNA transcription vector of claims  11  or the DNA vector of any of claims  12 - 18  carrying an exogenous DNA fragment.  
     
     
         27 . The host cell of  claim 26  wherein the cell is an avian, a mammalian, a reptilian, an amphibian, an insecticidal or a fish cell.  
     
     
         28 . The host cell of  claim 27  which is the hamster BHK cell.  
     
     
         29 . A method to produce the transformed host cell of  claim 26 ,  27  or  28  comprising transfection of the cell with the RNA of any of claims  1 - 8 , with the cDNA of  claim 11  or of any of claims  12 - 18  carrying an exogenous DNA fragment or infection of the cell with the infectious viral particles of  claim 9  or  10 .  
     
     
         30 . The method of  claim 29  wherein the transfection is produced by electroporation of the host cell.  
     
     
         31 . A method for the production of a polypeptide or protein comprising infection of animal host cells with infectious particles according to  claim 9  or  10 , containing exogenous RNA encoding said polypeptide or protein and produced according to method of  claim 20  or  21 , culturing the said transformed cells to express the exogenous RNA and isolation and purification of the product formed by said expression.  
     
     
         32 . A method for the production of a polypeptide or protein comprising in vitro transcription of the cDNA of the vector of any of claims  11 - 18  carrying an exogenous DNA fragment coding for the polypeptide or protein, transfection of animal host cells with the produced RNA transcript, transformed animal host cells being obtained harbouring the RNA transcript, culturing the said transformed cells to express the exogenous RNA and isolation and purification of the product formed by said expression.  
     
     
         33 . The metod of  claim 32  wherein the vector cDNA is comprised of the cDNA of the vector of  claim 17  carrying the exogenous DNA fragment.  
     
     
         34 . An antigen consisting of a chimaeric alphavirus having an exogenous epitopic peptide sequence or antigenic determinant inserted into its structural proteins.  
     
     
         35 . The antigen of  claim 34  wherein the chimaeric alpha-virus is derived from SFV.  
     
     
         36 . The antigen of  claim 34  or  35 , wherein the exogenous epitopic peptide sequence is comprised of an epitopic peptide sequence derived from a structural protein of a virus belonging to the immunodeficiency virus class inclusive of the human immunodeficiency virus types.  
     
     
         37 . Vaccine preparation comprising the antigen of  claim 34 ,  35  or  36  as immunizing component.  
     
     
         38 . Vaccine of  claim 37  wherein the chimaeric alphavirus is attenuated by comprising the conditionally lethal SFV mutation of  claim 17 , an amber (stop codon) a temperature sensitive mutation or other mutation in its genome.  
     
     
         39 . A method for the production of an antigen of claim  34 ,  35  or  36  comprising 
 a) in vitro transcription of the cDNA of the vector of any of claims  11 - 18  carrying an exogenous DNA fragment encoding the foreign epitopic peptide sequence or antigenic determinant and transfection of animal host cells with the produced RNA transcript, or  
 b) transfection of animal host cells with the said cDNA of the above step a), culturing the transfected cells and recovering the chimaeric alphavirus antigen.  
 
     
     
         40 . The method of  claim 32 ,  33  or  39  wherein the transfection is produced by electroporation of the host cell.  
     
     
         41 . A method for the production of an antigen in an organism by using in vivo infection with infectious particles according to  claim 9  or  10  containing exogenous RNA encoding an exogenous epitopic peptide sequence or antigenic determinant, and produced according the  claim 20  or  21 .