Stable recombinant MVA vectors comprising modified RSV genes with reduced intramolecular recombinatorial activity
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Publication Number
US-11225673-B2
Publication Date
2022-01-18
Expiration Date
Abstract
The invention relates to vectors comprising two or more homologous nucleotide sequences and methods for generating them. The invention concerns substituting bases in the homologous nucleotide sequences with different bases that do not alter the encoded amino acid sequence. The invention allows for the reduction of intramolecular recombination between homologous nucleotide sequences, in particular in mammalian cells. The invention further relates to nucleotide sequences containing substituted bases.
Core Innovation
The invention relates to stabilizing a recombinant modified vaccinia Ankara (MVA) virus vector that stably encodes homologous sequences. The strategy introduces synonymous nucleotide substitutions so that homologous sequences remain protein-identical while reducing intramolecular recombination mediated by homologous nucleotide identity.
The vector includes first and second nucleotide sequences of at least 1500 nucleotides each, each coding for at least 500 amino acids, with at least 150 continuous amino acids in each sequence having at least 75% amino acid identity. At least one of the first and second nucleotides includes at least 400 substituted nucleotides that do not alter identical amino acids encoded by the two nucleotide sequences, and the sequences differ by at least 400 nucleotides while sharing stretches of identity of no more than 9 contiguous nucleotides.
The concept is exemplified with recombinant MVA-BN constructs encoding RSV-F proteins, including RSV-F full-length and truncated RSV-F_trunc form. Substituted, truncated constructs are reported to be expressed and to yield stable double recombinants with no detectable recombination by PCR, and the strategy is described as reducing long identical nucleotide stretches while maintaining expression.
Codon-substitution is also applied to EBOV GP genes (EBOV-B, EBOV-S, and EBOV-Z), where expression is maintained and reduced long identical nucleotide stretches are reported.
Claims Coverage
The independent claim defines a recombinant MVA virus vector that stably encodes homologous sequences via two specified nucleotide sequences that preserve identical amino acids while substantially reducing contiguous nucleotide identity. The independent claim features are tied to length and identity constraints, synonymous substitutions, differences between the two nucleotide sequences, and a limit on contiguous stretches of identity, with the restriction that each nucleotide sequence encodes an RSV protein.
Stably encoding homologous sequences in recombinant MVA with constrained contiguous nucleotide identity
A recombinant MVA virus vector that stably encodes homologous sequences via first and second nucleotide sequences of at least 1500 nucleotides each, each coding for at least 500 amino acids, with at least 150 continuous amino acids encoded having at least 75% amino acid identity, wherein the first and second nucleotide sequences share stretches of identity of no more than 9 contiguous nucleotides.
Synonymous nucleotide substitutions preserving identical amino acids
At least one of the first and second nucleotides has at least 400 substituted nucleotides that do not alter the identical amino acids encoded by the two nucleotide sequences.
Substantial sequence difference while maintaining homologous protein identity
The first and second nucleotide sequences differ by at least 400 nucleotides while each encodes an RSV protein.
Encoding full-length and truncated RSV-F protein forms as the two homologous sequences
The recombinant MVA virus vector is defined such that the first and second nucleotide sequences encode a full-length RSV-F protein and a truncated RSV-F protein.
Encoding specified RSV-F amino acid sequences by SEQ ID
The recombinant MVA virus vector is defined such that its first and second nucleotide sequences encode the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4, respectively.
Encoding specified RSV-F nucleotide sequences by SEQ ID
The recombinant MVA virus vector is defined such that its first and second nucleotide sequences correspond to SEQ ID NO:1 and SEQ ID NO:2, respectively.
Overall claim coverage centers on a recombinant MVA virus vector that stably encodes homologous sequences by using two long nucleotide sequences that preserve homologous amino acid identity through synonymous substitutions, substantially differ at the nucleotide level, and limit shared contiguous nucleotide stretches to no more than 9 nucleotides, with the restriction that both sequences encode RSV protein; dependent refinements specify full-length versus truncated RSV-F and the particular SEQ ID protein and nucleotide identities.
Stated Advantages
Stabilizes recombinant MVA constructs that stably encode homologous sequences by reducing intramolecular recombination.
Maintains expression of RSV-F proteins in substituted, truncated recombinant constructs.
Produces stable double recombinants with no detectable recombination by PCR.
Maintains expression for recombinant MVA-BN constructs encoding EBOV GP genes while reducing long identical nucleotide stretches.
Documented Applications
Recombinant MVA-BN constructs expressing RSV-F protein variants (RSV-F full-length and RSV-F_trunc) as stable double recombinants with no detectable recombination by PCR.
Recombinant MVA-BN expressing three EBOV GP genes (EBOV-B, EBOV-S, and EBOV-Z) with maintained expression and reduced long identical nucleotide stretches.
Plasmid constructs containing RSV-F variants (RSV-F + RSV-F_trunc) referenced for plasmid stability in E. coli (plasmid pMISC210; stability described at a conceptual level in the provided content).
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