Intergenic sites between conserved genes in the genome of modified vaccinia ankara (MVA) vaccinia virus
Inventors
Moss, Bernard • Wyatt, Linda S. • Earl, Patricia L.
Assignees
US Department of Health and Human Services
Publication Number
US-10421978-B2
Publication Date
2019-09-24
Expiration Date
2027-08-24
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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.
Core Innovation
The invention provides new insertion sites for the integration of exogenous DNA sequences into the genome of modified vaccinia Ankara (MVA) virus, specifically in intergenic regions (IGRs) located between or flanked by two adjacent open reading frames (ORFs) of the vaccinia virus genome that correspond to conserved genes. It further provides related plasmid vectors useful for inserting exogenous DNA into the vaccinia virus genome, and recombinant vaccinia viruses comprising such exogenous sequences inserted at these new sites for use as medicines or vaccines.
The problem being solved relates to the instability of recombinant MVAs with heterologous DNA sequences inserted at traditional sites, which can undergo deletions or mutations due to non-essential sequences being lost during virus replication. Prior recombinant MVA viruses sometimes exhibited instability, especially when foreign genes were inserted into naturally occurring deletion sites, leading to deletions and loss of inserted gene expression. The inventors addressed the problem by inserting heterologous DNA between conserved essential genes such that deletions compromising these essential regions would be non-viable, thereby producing more stable recombinant viruses.
Despite the intergenic regions potentially containing regulatory elements crucial for transcriptional control, the inventors demonstrated that inserting exogenous sequences into these IGRs does not alter typical characteristics or gene expression of MVA. By selecting IGRs flanked by pairs of conserved essential genes, especially those with specific gene orientations (such as "end to end" where stop codons of adjacent genes are close), the invention enables stable exogenous sequence insertion without disrupting viral function. The invention also includes methods of coding sequence modification (such as silent mutations to reduce hot spots prone to mutation) and spacer sequences to enhance stability of insertions.
Claims Coverage
The patent claims focus on plasmid vectors, insertion methods, and recombinant vaccinia viruses involving heterologous DNA sequences inserted between adjacent essential ORFs in the vaccinia virus genome, with emphasis on vectors capable of homologous recombination with MVA genomes.
Plasmid vector for insertion between essential adjacent ORFs
A plasmid vector comprising a cloning site flanked by nucleic acid sequences derived from the 3′ termini of two adjacent, essential open reading frames (ORFs) oriented 3′ end-to-3′ end in the vaccinia virus genome, capable of homologous recombination with an MVA virus genome.
Inclusion of heterologous DNA sequence in the plasmid vector
The plasmid vector comprises a heterologous DNA sequence inserted between the two nucleic acid sequences derived from the adjacent essential ORFs.
Modification of inserted heterologous DNA to reduce mutation hot spots
The inserted heterologous DNA sequence is modified by identifying runs of four or more contiguous guanine (G) or cytosine (C) residues and making silent mutations to reduce these runs to less than four, enhancing insertion stability.
Applicability to specific MVA strains
The plasmid vector and methods apply to vaccinia viruses including MVA deposited as ATCC PTA-5095 and MVA sequences from GenBank accession numbers AY603355 and U94848.
Selection of specific adjacent ORFs for insertion sites
The two adjacent ORFs flanking the insertion site are selected from a defined group of essential gene pairs, including but not limited to I8R-G1L and several other pairs enumerated with reference to GenBank AY603355.
Method for producing recombinant vaccinia virus with heterologous DNA between essential ORFs
A method involving transfection of a cell with the plasmid vector containing the heterologous DNA placed between nucleic acid sequences derived from adjacent essential ORFs, followed by infection of the transfected cell with vaccinia virus to produce recombinant virus.
Plasmid vector comprising an intergenic region flanked by essential ORFs
A plasmid vector comprising an intergenic region (IGR) flanked by nucleic acid sequences derived from proximal ends of two adjacent essential ORFs in vaccinia virus genome, capable of homologous recombination with MVA genome, with capacity to include heterologous DNA sequence in the IGR.
The claims cover plasmid vectors and methods for stable insertion of heterologous DNA sequences into MVA genomes at new intergenic sites between conserved essential ORFs, including specific modifications to reduce mutation and choice of essential gene pairs to ensure stability and viability of recombinant viruses.
Stated Advantages
Insertion of exogenous DNA sequences into intergenic regions between conserved essential genes allows stable integration without altering MVA characteristics or gene expression.
Inserting heterologous genes between essential genes prevents deletions of the insert due to virus replication advantage, enhancing stability during viral propagation and large-scale amplification.
Use of silent mutations to reduce runs of contiguous G or C nucleotides in the inserted DNA sequences decreases mutation hot spots and stabilizes gene expression.
The new insertion sites avoid disruption of coding sequences and promoter elements, thereby preserving viral viability and replication competence.
The approach facilitates production of recombinant vaccinia viruses for clinical use, allowing consistent expression of foreign antigens such as HIV genes.
Documented Applications
Use of recombinant MVA viruses with heterologous sequences inserted at the new sites as medicines or vaccines, including vaccines against infectious diseases like HIV.
Immunization regimens employing recombinant MVA vectors encoding HIV antigens to prime and boost CD8+ T cell immune responses and antibody responses in mammals including humans.
Production of pharmaceutical compositions and vaccines comprising recombinant MVA viruses for prophylactic or therapeutic vaccination.
In vitro and in vivo gene delivery methods wherein recombinant MVA viruses deliver exogenous coding sequences encoding therapeutic proteins, antigens, or markers.
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