Intergenic sites between conserved genes in the genome of modified vaccinia ankara (mva) vaccinia virus
AssigneeTHE USA, as represented by the Secretary, Departme
InventorsBernard Moss, Linda Wyatt, Patricia Earl
Life of this patent
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Abstract
The present invention relates to new insertion sites useful for the integration of exogenous sequences 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 where 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.
Claims33
A recombinant modified vaccinia Ankara (MVA) virus comprising a heterologous DNA sequence inserted into an intergenic region (IGR) of the MVA genome, wherein the IGR is located between or is flanked by two adjacent open reading frames (ORFs) of the MVA genome, and wherein the ORFs correspond to conserved genes, and wherein the two adjacent ORFs are selected from the group consisting of (using the nomenclature according to CDC/Acambis):
- 044-045, 049-050, 050-051, 058-059, 064-065, 065-066, 069-070, 070-071, 071-072, 072-073, 073-074, 074-075, 075-076, 076-077, 077-078, 078-079, 081-082, 085-086, 086-087, 089-090, 092-093, 093-094, 095-096, 097-098, 100-101, 101-102, 102-103, 104-105, 108-109, 113-114, 114-115, 118-119, 121-122, 122-123, 123-124, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 141-142, and 142-143, in an exemplary manner or corresponding thereto in other strains of vaccinia virus.
A plasmid vector comprising a DNA sequence derived from or homologous to the genome of an MVA, wherein said DNA sequence comprises a complete or partial fragment of an IGR sequence located between or flanked by two adjacent ORFs of the MVA genome and inserted into said IGR sequence a cloning site for the insertion of a DNA sequence being heterologous to the genome of the MVA and, optionally, of a reporter- and/or selection gene cassette, and wherein the two adjacent ORFs are selected from the group consisting of (using the nomenclature according to CDC/Acambis):
- 044-045, 049-050, 050-051, 058-059, 064-065, 065-066, 069-070, 070-071, 071-072, 072-073, 073-074, 074-075, 075-076, 076-077, 077-078, 078-079, 081-082, 085-086, 086-087, 089-090, 092-093, 093-094, 095-096, 097-098, 100-101, 101-102, 102-103, 104-105, 108-109, 113-114, 114-115, 118-119, 121-122, 122-123, 123-124, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 141-142, and 142-143, in an exemplary manner or corresponding thereto in other strains of vaccinia virus.
A plasmid vector comprising a DNA sequence derived from or homologous to the genome of an MVA, wherein said DNA sequence comprises IGR flanking sequences of two adjacent ORFs and, inserted into said IGR, a cloning site for the insertion of a DNA sequence being heterologous to the genome of the MVA and, optionally, of a reporter- and/or selection gene cassette, and wherein the two adjacent ORFs are selected from the group consisting of (using the nomenclature according to CDC/Acambis):
- 044-045, 049-050, 050-051, 058-059, 064-065, 065-066, 069-070, 070-071, 071-072, 072-073, 073-074, 074-075, 075-076, 076-077, 077-078, 078-079, 081-082, 085-086, 086-087, 089-090, 092-093, 093-094, 095-096, 097-098, 100-101, 101-102, 102-103, 104-105, 108-109, 113-114, 114-115, 118-119, 121-122, 122-123, 123-124, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 141-142, and 142-143, in an exemplary manner or corresponding thereto in other strains of vaccinia virus.
A method for producing the MVA according to claim 1 comprising transfecting a cell with a plasmid vector according to any of claims 21 to 39;
- infecting the transfected cell with an MVA;
- and identifying, isolating and, optionally, purifying the MVA.
A method of stabilizing an insert in a recombinant vaccinia virus comprising identifying a run of 4 or more G or C residues in said insert, and making a silent mutation in said run so identified by substitution so that the length of the run is reduced.
pLW-73.
MVA/UGD4d.
A recombinant vaccinia virus comprising a heterologous DNA sequence inserted into an intergenic region (IGR) of the vaccinia virus genome, wherein 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 wherein the two adjacent ORFs are selected from the group consisting of (using the nomenclature according to CDC/Acambis):
- 044-045, 049-050, 050-051, 058-059, 064-065, 065-066, 069-070, 070-071, 071-072, 072-073, 073-074, 074-075, 075-076, 076-077, 077-078, 078-079, 081-082, 085-086, 086-087, 089-090, 092-093, 093-094, 095-096, 097-098, 100-101, 101-102, 102-103, 104-105, 108-109, 113-114, 114-115, 118-119, 121-122, 122-123, 123-124, 127-128, 128-129, 129-130, 130-131, 131-132, 132-133, 133-134, 134-135, 135-136, 141-142, and 142-143, in an exemplary manner or corresponding thereto in other strains of vaccinia virus.
Dependencies are inferred from the claim text.