High throughput assay for measuring adenovirus replication kinetics

Abstract

Recombinant adenovirus genomes that include a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence are described. The recombinant adenovirus genomes and recombinant adenoviruses produced by the disclosed genomes can be used, for example, in high-throughput assays to measure virus replication kinetics. Methods for measuring replication kinetics of a recombinant adenovirus are also described.

1 . A recombinant adenovirus genome, comprising a heterologous open reading frame (ORF) and a self-cleaving peptide coding sequence, both operably linked to and in the same reading frame as an endogenous adenovirus ORF, wherein the self-cleaving peptide coding sequence is located between the heterologous ORF and the endogenous ORF, and wherein:
 the endogenous ORF is E1B-55k and the heterologous ORF is 3′ of E1B-55k;   the endogenous ORF is DNA polymerase and the heterologous ORF is 5′ of DNA polymerase;   the endogenous ORF is DNA-binding protein (DBP) and the heterologous ORF is 3′ of DBP;   the endogenous ORF is adenovirus death protein (ADP) and the heterologous ORF is 5′ of ADP;   the endogenous ORF is E3-14.7k and the heterologous ORF is 3′ of E3-14.7k; or   the endogenous ORF is E4-ORF2 and the heterologous ORF is 5′ of E4-ORF2.   
     
     
         2 . The recombinant adenovirus genome of  claim 1 , wherein the self-cleaving peptide is a 2A peptide or variant thereof. 
     
     
         3 . The recombinant adenovirus genome of  claim 2 , wherein the 2A peptide comprises a porcine teschovirus-1 (PTV1) 2A (P2A) peptide, a foot and mouth disease virus (FMDV) 2A (F2A) peptide, an equine rhinitis A virus (ERAV) 2A (E2A) peptide or a Thosea asigna virus (TaV) 2A (T2A) peptide, or a variant thereof. 
     
     
         4 . The recombinant adenovirus genome of  claim 3 , wherein the amino acid sequence of the self-cleaving peptide is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 12-19. 
     
     
         5 . The recombinant adenovirus genome of  claim 3 , wherein the self-cleaving peptide comprises the amino acid sequence of any one of SEQ ID NOs: 12-19. 
     
     
         6 . The recombinant adenovirus genome of  claim 1 , wherein the heterologous ORF encodes a fluorescent protein. 
     
     
         7 . The recombinant adenovirus genome of  claim 6 , wherein the fluorescent protein is a green fluorescent protein (GFP) a yellow fluorescent protein (YFP), a red fluorescent protein (RFP) or a blue fluorescent protein (BFP). 
     
     
         8 . The recombinant adenovirus genome of  claim 7 , wherein the YFP is YPet or the RFP is mCherry. 
     
     
         9 . The recombinant adenovirus genome of  claim 1 , comprising in the 5′ to 3′ direction:
 E1B-55K-P2A-YPet; 
 E1B-55K-P2A-mCherry; 
 YPet-P2A-(DNA polymerase); 
 DBP-P2A-YPet; 
 YPet-P2A-ADP; 
 E3-14.7k-P2A-YPet; 
 YPet-P2A-E4-ORF2; or 
 mCherry-P2A-E4-ORF2. 
 
     
     
         10 . The recombinant adenovirus genome of  claim 1 , comprising the nucleotide sequence of any one of SEQ ID NOs: 3-7, 9-11, 20 and 21. 
     
     
         11 . A recombinant adenovirus comprising the recombinant adenovirus genome of  claim 1 . 
     
     
         12 . A kit comprising:
 (i) the recombinant adenovirus genome of  claim 1 ; and   (ii) cells, cell culture media and/or a multi-well plate.