Intergenic Sites Between Conserved Genes in the Genome of Modified Vaccinia Ankara (MVA) Vaccinia Virus

Abstract

The pesent invention relates to new insertion sites useful for the integration exogenous sequence into an intergenic region (IGR) of a vaccinia virus genome, where the IGR is located between or is flanked by two adjacent open reading frames (ORFs) of the vaccinia virus genome, and wherein the ORFs correspond to conserved genes, and to related plasmid vectors useful to insert exogenous DNA into the genome of a vaccinia virus, and further to recombinant vaccinia viruses comprising an exogenous sequence inserted into said new insertion site as a medicine or vaccine.

1 - 33 . (canceled) 
     
     
         34 . A method of stabilizing a heterologous nucleic acid insert in a recombinant virus, the method comprising making a mutation in a run of 4 or more purines or 4 or more pyrimidines, in the nucleic acid insert, so that the length of the run of the 4 or more purines or the 4 or more pyrimidines is reduced. 
     
     
         35 . The method of  claim 34 , wherein the mutation is a substitution mutation. 
     
     
         36 . The method of  claim 34 , wherein the mutation is a silent mutation. 
     
     
         37 . The method of  claim 34 , wherein the mutation is made in a run of 4 or more guanine (G) or cytosine (C) residues. 
     
     
         38 . The method of  claim 34 , wherein the mutation is made in a run of 4 or more adenine (A) or thymine (T) residues. 
     
     
         39 . The method of  claim 34 , wherein the recombinant virus is a recombinant vaccinia virus. 
     
     
         40 . The method of  claim 39 , wherein the heterologous nucleic acid insert is located in an intergenic region (IGR) of the vaccinia virus (VV) genome, wherein the IGR is located between or is flanked by two adjacent open reading frames (ORFs) of the VV genome, and wherein the ORFs correspond to genes that are essential for replication of vaccinia virus. 
     
     
         41 . The method of  claim 34 , wherein the sequence of the heterologous nucleic acid insert is derived from human immunodeficiency virus (HIV). 
     
     
         42 . The method of  claim 34 , wherein the heterologous nucleic acid insert comprises the HIV gagpol gene or the HIV env gene. 
     
     
         43 . A recombinant virus comprising a heterologous nucleic acid insert, wherein the presence of the heterologous insert has been stabilized using the method of  claim 34 . 
     
     
         44 . The recombinant virus of  claim 43 , wherein the virus is a vaccinia virus. 
     
     
         45 . The recombinant virus of  claim 44 , wherein the heterologous nucleic acid insert is located in an intergenic region (IGR) of the vaccinia virus (VV) genome, wherein the IGR is located between or is flanked by two adjacent open reading frames (ORFs) of the VV genome, and wherein the ORFs correspond to genes that are essential for replication of vaccinia virus. 
     
     
         46 . The recombinant virus of  claim 43 , wherein the sequence of the heterologous nucleic acid insert is derived from human immunodeficiency virus (HIV). 
     
     
         47 . The recombinant virus of  claim 43 , wherein the heterologous nucleic acid insert comprises the HIV gagpol gene or the HIV env gene.