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-9879231-B2
Publication Date
2018-01-30
Expiration Date
2030-10-15
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 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) virus containing restructured sites that are 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. In the recombinant MVA virus, these essential ORFs are made adjacent even though they are non-adjacent in the parental MVA virus used to construct the recombinant virus. This structural rearrangement provides a new, stable site for insertion of heterologous nucleic acid sequences.
The problem being solved is that recombinant MVA viruses often exhibit instability of inserted heterologous DNA sequences, resulting in loss or mutation of the foreign gene after tissue culture passage. This instability can reduce expression of the foreign gene and compromise the immunogenicity or utility of the recombinant virus as a vaccine vector. Previous heterologous insertions in non-essential regions of the MVA genome were prone to deletion or mutation, which gave a growth advantage to mutants lacking the foreign gene, leading to their overgrowth and instability of recombinant MVA populations.
The invention addresses this problem by removing non-essential DNA regions and restructuring the viral genome so that two essential ORFs, originally separated by non-essential ORFs, are made adjacent in the recombinant MVA virus. Because deletion of essential genes causes a growth disadvantage for the virus, any deletion including essential ORFs is selected against, thereby stabilizing the foreign gene insertion located in the intervening intergenic region. This method improves the genetic stability of recombinant MVA viruses containing inserted heterologous sequences encoding therapeutically useful proteins, such as antigens.
Claims Coverage
The patent includes independent claims directed to isolated nucleic acid constructs comprising adjacent essential open reading frames (ORFs) from a poxvirus genome and methods of producing stable recombinant viruses using such constructs. The main inventive features relate to restructuring viral genomes and insertion of heterologous sequences between essential ORFs that are non-adjacent in parental viruses.
Nucleic acid constructs with adjacent essential ORFs separated by removed non-essential ORFs
Comprise a first and second nucleic acid sequence derived from or homologous to essential poxvirus ORFs that are separated by at least one non-essential ORF in the parental poxvirus genome, but are adjacent to each other in the nucleic acid construct.
Inclusion of heterologous sequences and intergenic regions between essential ORFs
The nucleic acid constructs may include an intergenic region or a heterologous nucleic acid sequence between the two adjacent essential ORF nucleic acid sequences, enabling insertion of foreign genes under transcriptional control elements.
Selection of essential ORFs from specific groups for stable insertion sites
The essential ORFs used in the constructs and recombinant viruses are selected from a defined group including A50R, B1R, F10, F12, F13L, F15L, F17R, E4L, E6L, E8L, E10L, I1L, I3L, I5L, J1R, J3R, D7L, D9L, A24R, and A28R. Pairs used to flank insertions include, among others, A50R-B1R.
Method of producing stable recombinant poxvirus using constructs
Involves transfecting cells with the nucleic acid construct comprising adjacent essential ORFs as above, infecting these cells with poxvirus, and culturing under conditions that allow homologous recombination, resulting in recombinant poxviruses with restructured genomes and stable insertions.
The claims cover nucleic acid constructs and methods for producing recombinant poxviruses in which heterologous nucleic acid sequences are inserted into intergenic regions between essential ORFs that have been made adjacent by exclusion of non-essential ORFs present in parental viruses, providing enhanced genetic stability of inserted sequences.
Stated Advantages
Insertion of heterologous genes between adjacent essential ORFs stabilizes the recombinant MVA by preventing deletions that include essential genes, thus maintaining foreign gene integrity during virus replication and passage.
Codon alteration of heterologous genes at mutation hot spots further enhances stability by preventing point mutations that cause premature termination of translation.
The restructured genome maintains virus growth characteristics and gene expression profile, ensuring functional recombinant viruses suitable for vaccine and therapeutic applications.
The approach allows production of large-scale vaccine seed stocks with stable foreign gene expression, important for clinical and commercial vaccine development.
Documented Applications
Use of recombinant MVA viruses containing stable heterologous sequences as vaccines, particularly vaccines against infectious diseases such as HIV/AIDS.
Vaccination regimens using recombinant MVA vectors to prime or boost CD8+ T cell immune responses and elicit antibody responses against antigens encoded by inserted heterologous genes.
Production of recombinant viruses expressing therapeutically interesting genes to induce immune responses in mammals including immune-compromised individuals.
Use of recombinant MVA viruses for introducing heterologous nucleic acid coding sequences into target cells for production of proteins, antigens, or therapeutic molecules.
Interested in licensing this patent?