Modified vaccinia Ankara (MVA) virus recombinants comprising heterologous coding sequences inserted into the intergenic regions between essential genes
Inventors
Moss, Bernard • Wyatt, Linda • Earl, Patricia
Assignees
US Department of Health and Human Services
Publication Number
US-9133478-B2
Publication Date
2015-09-15
Expiration Date
2027-08-24
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 relates to new insertion sites for integration of exogenous DNA sequences into intergenic regions (IGRs) of the modified vaccinia Ankara (MVA) virus genome. These IGRs are located between or flanked by two adjacent open reading frames (ORFs) of the MVA genome, where the ORFs correspond to conserved or essential genes for vaccinia virus replication. The invention also includes related plasmid vectors for inserting exogenous DNA into the vaccinia virus genome and recombinant vaccinia viruses comprising exogenous sequences inserted into these new insertion sites for use as medicines or vaccines.
The problem addressed is that insertion of foreign genes into the MVA genome at common sites has led to instability of recombinant viruses due to deletions or mutations of the inserted genes. The MVA genome tends to delete nonessential sequences during propagation, and insertions at natural deletion sites sometimes result in unstable recombinants. The inventors sought insertion sites between conserved essential genes where deletions would be deleterious to the virus, thereby ensuring stable retention of exogenous DNA.
A further innovation is the discovery and construction of plasmid vectors that direct insertion of foreign genes precisely into these intergenic regions flanked by essential genes, such as the I8R-G1L locus, to prevent selection of deletion mutants. In addition, the inventors identified "hot spots" prone to point mutations leading to gene instability and applied codon alteration strategies to stabilize the inserted sequences. The recombinant MVA containing exogenous DNA in these sites retain typical viral gene expression and characteristics without affecting essential MVA functions.
Claims Coverage
The patent claims sixteen main inventive features across multiple independent claims covering recombinant MVA viruses, insertion sites, DNA sequences, methods, and compositions.
Recombinant MVA with heterologous DNA in essential gene intergenic region
A recombinant modified vaccinia Ankara (MVA) virus comprising a heterologous DNA sequence inserted into an intergenic region (IGR) between two adjacent ORFs of the MVA genome, wherein the ORFs correspond to genes essential for vaccinia virus replication.
Selection of specific essential gene pairs for insertion
The two adjacent ORFs flanking the inserted heterologous sequence are selected from specific essential gene pairs such as I8R-G1L, F17R-E1L, G2R-G4L, and others as listed according to GenBank accession number AY603355.
Insertion of heterologous DNA under poxviral promoter control
The heterologous DNA sequence is operably linked to a poxviral transcription control element enabling expression within the MVA genome.
Encoding of proteins, peptides, or polypeptides by heterologous DNA
The heterologous DNA encodes one or more proteins, polypeptides, or peptides, including but not limited to sequences derived from HIV such as HIV Env.
Use of MVA strains with known genomic sequences
Application of the insertion in MVA genomes corresponding to deposited strains, including ATCC deposit PTA-5095 and GenBank accession numbers AY603355 and U94848.
Immunogenic and pharmaceutical compositions comprising recombinant MVA
Vaccines and pharmaceutical compositions comprising the recombinant MVA viruses with exogenous inserted sequences.
Methods of inducing immune responses and protein production
Methods for inducing immune responses in animals by administering recombinant MVA, and methods for producing proteins or peptides by infecting host cells with the recombinant MVA expressing heterologous DNA.
Stabilization via codon alteration of hot spots
Modification of inserted heterologous DNA sequences by silent mutations to reduce consecutive identical G or C nucleotide runs, stabilizing the gene against point mutations.
The claims cover recombinant MVA viruses with heterologous DNA stably inserted into intergenic regions between essential vaccinia genes, specifying particular gene pairs, with expression controlled by poxviral promoters. The claims also include compositions, methods of use, and stabilization techniques for maintaining gene integrity during viral replication and passage.
Stated Advantages
The new insertion sites allow stable integration of exogenous DNA without disrupting essential vaccinia virus genes or viral functions.
Insertion between essential genes prevents deletion mutants from replicating, thereby maintaining stable expression of foreign genes.
Codon alteration of mutation-prone sequences further stabilizes inserted genes against point mutations during viral propagation.
The recombinant viruses preserve the typical characteristics and gene expression profiles of MVA.
The method facilitates production of stable recombinant MVA for vaccines or medicines, especially in clinical trial scale-up.
Documented Applications
Use as recombinant vaccines or medicines expressing heterologous antigens, especially HIV antigens such as Env and GagPol proteins.
Inducing and boosting CD8+ T cell immune responses and antibody responses against HIV antigens in animals and humans.
Production of proteins, polypeptides, or peptides in host cells infected with recombinant MVA.
Use in prime and boost immunization protocols involving DNA vaccines, adenovirus vectors, and recombinant MVA to enhance immune protection.
Interested in licensing this patent?