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US 10,059,925 B2Granted
High titer recombinant influenza viruses with enhanced replication in vero cells
AssigneeWisconsin Alumni Research Foundation (WARF)
InventorsYoshihiro Kawaoka, Taisuke Horimoto, Shin Murakami
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Abstract
The invention provides a composition useful to prepare high titer influenza viruses, e.g., in the absence of helper virus, which includes internal genes from an influenza virus vaccine strain or isolate, e.g., one that is safe in humans, for instance, one that does not result in significant disease, and genes from vaccine seed virus isolates which include a HA gene segment with a HA2 sequence encoding a HA2 that confers enhanced growth in cells in culture, such as Vero cells.
Claims20
A method to prepare an influenza virus with enhanced replication in Vero cells, comprising:
- providing a vector comprising a recombinant nucleic acid molecule comprising sequences for an influenza virus HA segment from a first influenza virus isolate, which segment encodes an HA with an amino acid other than aspartic acid or glutamic acid at position 117 in HA2, wherein the numbering for HA2 residues is that for H1 HA2;
- modifying the HA segment to encode an aspartic acid or glutamic acid at position 117 in HA2, thereby yielding a modified HA segment;
- and
- contacting a cell with a vector comprising promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus PA segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus PB1 segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus PB2 segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to the modified HA segment linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus NP segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus NA segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus M segment DNA linked to a transcription termination sequence, and a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus NS segment DNA linked to a transcription termination sequence;
- and
- a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus M2, or a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NS1 or a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NS2;
- in an amount effective to yield infectious influenza virus.
4 more elements
A method to prepare an influenza virus with enhanced replication in Vero cells, comprising:
- providing a vector comprising a recombinant nucleic acid molecule comprising sequences for an influenza virus HA segment from a first influenza virus isolate, which segment is modified to encode an HA with aspartic acid or glutamic acid at position 117 in HA2, wherein the numbering for HA2 residues is that for H1 HA2;
- and
- contacting a cell with a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus PA segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus PB1 segment DNA linked to a transcription termination sequence, a vector for comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus PB2 segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to the modified HA segment linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus NP segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus NA segment DNA linked to a transcription termination sequence, a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus M segment DNA linked to a transcription termination sequence, and a vector comprising a promoter that yields full length, genomic influenza virus RNA or its complement operably linked to an influenza virus NS segment DNA linked to a transcription termination sequence;
- and
- a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus PB2, and a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NP, and optionally a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus HA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NA, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus M1, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus M2, a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NS1 or a vector comprising a promoter that yields mRNA operably linked to a DNA segment encoding influenza virus NS2,
- in an amount effective to yield infectious influenza virus.
3 more elements
A method to prepare an influenza virus with enhanced replication in Vero cells, comprising:
- providing an isolated influenza virus having a PA segment, a PB1 segment, a PB2 segment, a NP segment, a NS segment, a M segment, a NA segment, and a HA segment;
- and
- modifying the HA segment to encode an aspartic acid or glutamic acid at position 117 in HA2, wherein the numbering for HA2 residues is that for H1 HA2, thereby yielding a modified influenza virus.
Dependencies are inferred from the claim text.