Recombinant modified vaccinia ankara (MVA) vaccinia virus containing restructured insertion sites
Inventors
Moss, Bernard • Wyatt, Linda S. • Earl, Patricia L.
Assignees
US Department of Health and Human Services
Publication Number
US-9133480-B2
Publication Date
2015-09-15
Expiration Date
2030-10-15
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Abstract
The present invention relates to recombinant modified vaccinia Ankara (MVA) virus containing restructured sites useful for the integration of heterologous nucleic acid sequences into an intergenic region (IGR) of the virus genome, where the IGR is located between two adjacent, essential open reading frames (ORFs) of the vaccinia virus genome, wherein the adjacent essential ORFs are non-adjacent in a parental MVA virus used to construct the recombinant MVA virus, and to related nucleic acid constructs useful for inserting heterologous DNA into the genome of a vaccinia virus, and further to the use of the disclosed viruses as a medicine or vaccine.
Core Innovation
The invention relates to recombinant modified vaccinia Ankara (MVA) viruses whose genomes have been restructured by removing non-essential open reading frames (ORFs) located between two adjacent essential ORFs. By making the essential ORFs adjacent, heterologous nucleic acid sequences can be stably integrated into intergenic regions (IGRs) between these essential ORFs. These regions are stable since loss of essential genes confers a growth disadvantage to the virus, preventing the deletion of inserted heterologous sequences during virus propagation.
The problem addressed is the instability of heterologous DNA sequences inserted into the MVA genome, particularly when inserted into regions containing non-essential genes. Such insertions are often lost due to deletions, as viruses lacking heterologous genes and some flanking non-essential sequences have a growth advantage, leading to overgrowth of non-expressing mutants. This instability complicates the production of recombinant MVA viruses for vaccines, especially for large-scale seed stocks.
The invention provides nucleic acid constructs comprising adjacent essential ORFs from MVA that are non-adjacent in the parental virus but made adjacent in the construct to direct homologous recombination that deletes intervening non-essential ORFs in the viral genome. This restructuring creates stable insertion sites for heterologous nucleic acid sequences encoding therapeutically useful proteins, such as HIV antigens. Methods for producing such stable recombinant MVA viruses using these constructs are also disclosed, alongside uses of the recombinant viruses for prevention and treatment of disease.
Claims Coverage
The patent claims cover recombinant MVA viruses with heterologous nucleic acid sequences stably inserted between adjacent essential ORFs made adjacent by deletion of intervening non-essential ORFs, nucleic acid constructs enabling such recombinant viruses, and methods of producing and using said viruses.
Recombinant MVA virus with stable heterologous nucleic acid between adjacent essential ORFs
A recombinant MVA virus comprising a heterologous nucleic acid sequence located between two adjacent essential open reading frames of the MVA genome, wherein non-essential ORFs present between these essential ORFs in the parental MVA virus are deleted to make the essential ORFs adjacent in the recombinant virus.
Insertion of heterologous nucleic acid in intergenic regions
The heterologous nucleic acid is inserted in an intergenic region located between the two adjacent essential ORFs, preserving viral gene regulatory elements.
Use of ORF pairs flanking Del III insertion site
The recombinant MVA virus has non-essential ORFs flanking the Del III insertion site of parental MVA removed, with essential ORFs A50R and B1R made adjacent to flank the heterologous sequence.
Nucleic acid constructs for restructuring MVA genome
Isolated nucleic acid constructs comprising two essential ORF sequences from MVA that are non-adjacent in the parental genome but adjacent in the construct, enabling deletion of intervening non-essential ORFs during homologous recombination and stable insertion of heterologous sequences.
Method of producing recombinant MVA via homologous recombination
Methods involving transfecting cells with nucleic acid constructs containing adjacent essential ORF sequences flanking heterologous nucleic acid, infecting with parental MVA, culturing to allow recombination and isolation of stable recombinant MVA virus.
Uses of recombinant MVA virus
Methods for inducing immune responses or producing heterologous proteins by administering or infecting with the recombinant MVA virus containing heterologous nucleic acid encoding the protein of interest.
Selection of essential ORFs for adjacency
The two adjacent essential ORFs flank the heterologous sequence and are selected from multiple essential ORFs such as A50R, B1R, F10L, F12L, and others, with at least 90% sequence identity to wild-type sequences.
The claims define recombinant MVA viruses with heterologous sequences stably inserted between adjacent essential ORFs made adjacent by removal of intervening non-essential ORFs, nucleic acid constructs enabling such genome restructuring, methods for recombinant virus production by homologous recombination, and methods for immunization and protein production using such viruses.
Stated Advantages
Stable retention of heterologous genes by insertion between two essential vaccinia virus genes prevents deletion variants from overgrowing the virus population.
Improved genetic stability of inserted genes by altering mutation-prone nucleotide sequences through silent mutations.
Use of essential gene adjacency in the viral genome creates insertion sites that maintain inserted sequences during virus propagation and large-scale production.
Stable recombinant MVA viruses allow for reproducible large-scale manufacturing of vaccine candidates, including HIV vaccines.
Recombinant MVA viruses with removed non-essential genes and adjacency of essential ORFs exhibit stability of heterologous inserts over multiple passages.
Documented Applications
Use of recombinant MVA viruses containing heterologous sequences as vaccines for prevention and treatment of infectious diseases, exemplified by HIV.
Production of heterologous proteins in vitro by infection of host cells with recombinant MVA viruses containing coding sequences for such proteins.
Use in prime-boost immunization regimes to induce and boost CD8+ T cell and antibody responses against HIV antigens.
Generation of immune responses in living animals, including humans and non-human mammals, by direct administration of recombinant MVA vaccines.
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