Generation of infectious influenza viruses from virus-like particles
Compositions and methods to prepare influenza virus-like particles (VLPs) are provided.
1. A set of isolated host cells, comprising:
a first host cell for producing a first VLP with at least one and up to eight influenza A or B virus segments comprising:
one or more vectors which include at least one and up to eight transcription cassettes for production of at least one and up to eight viral segments from the genome of influenza A virus selected from:
a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus M DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NS DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NA DNA; or
a transcription cassette comprising a promoter operably linked to an influenza virus HA DNA or a non-influenza host cell binding protein DNA; or
one or more vectors which include at least one and up to eight transcription cassettes for production of at least one and up to eight viral segments from the genome of influenza B virus selected from:
a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus M DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NS DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NA and NB DNA; or a transcription cassette comprising a promoter operably linked to an influenza virus HA DNA or a non-influenza host cell binding protein DNA;
and
one or more vectors which include transcription cassettes for mRNA production including a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA; a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA; a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA; a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA; and optionally a transcription cassette comprising a promoter operably linked to an influenza virus HA or a non-influenza host cell binding protein
and
a second host cell for producing a second VLP with at least one and up to eight influenza A or B virus viral segments comprising:
one or more vectors which include at least one and up to eight transcription cassettes for production of at least one and up to eight viral segments from the genome of influenza A selected from:
a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus M DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NS DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NA DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus HA DNA or a non-influenza host cell binding protein DNA; or
one or more vectors which include at least one and up to eight transcription cassettes for production of at least one and up to eight viral segments from the genome of influenza B selected from:
a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus M DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NS DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NA and NB DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus HA DNA or a non-influenza host cell binding protein DNA;
and
one or more vectors which include transcription cassettes for mRNA production including a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA; a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA; a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA; and a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA; and optionally a transcription cassette comprising a promoter operably linked to an influenza virus HA or a non-influenza host cell binding protein,
wherein the first host cell has at least one transcription cassette for production of at least one viral segment that is not present in the second host cell or wherein the first host cell has at least one transcription cassette for production of at least one viral segment that is modified in the corresponding viral segment in the second host cell so that a functional influenza virus protein is not expressed from the corresponding segment.
2. The set of host cells of claim 1 wherein the second host cell has at least one transcription cassette for production of at least one viral segment that is not present in the first host cell.
3. The set of host cells of claim 1 wherein the first host cell has 7 of the expression cassettes for production of the viral segments or the second host cell has 7 of the expression cassettes for production of the viral segments; or wherein the first host cell has at least 2 of the expression cassettes for production of the viral segments and the second host cell has at least 6 of the expression cassettes for production of the viral segments; or wherein the first host cell has at least 3 of the expression cassettes for production of the viral segments and the second host cell has at least 5 of the expression cassettes for production of the viral segments; or wherein the first host cell has at least 4 of the expression cassettes for production of the viral segments and the second host cell has at least 4 of the expression cassettes for production of the viral segments; or wherein the first host cell has at least 5 of the expression cassettes for production of the viral segments and the second host cell has at least 3 of the expression cassettes for production of the viral segments; or wherein the first host cell has at least 6 of the expression cassettes for production of the viral segments and the second host cell has at least 2 of the expression cassettes for production of the viral segments.
4. The set of host cells of claim 1 wherein the first host cell has less than 7 of the expression cassettes for production of the viral segments or the second host cell has less than 7 of the expression cassettes for production of the viral segments.
5. The set of host cells of claim 1 wherein if the transcription cassettes for viral segments in the first and second host cells do not include all eight influenza A virus segments, the set further comprises one or more host cells that each produce a VLP with an influenza A virus segment that is not present in the first and second host cells.
6. The set of host cells of claim 1 wherein the first host cell, the second host cell or both the first and the second host cell stably expresses one or more of the transcription cassettes for mRNA production.
7. The set of host cells of claim 1 wherein the first or second host cell further comprises a transcription cassette comprising a promoter linked to 5′ influenza virus sequences comprising 5′ influenza virus noncoding sequences linked to a DNA of interest linked to 3′ influenza virus sequences comprising 3′ influenza virus noncoding sequences linked to a transcription termination sequence.
8. The set of host cells of claim 1 wherein in the first host cell if the one or more transcription cassettes for viral segments do not include a transcription cassette comprising a promoter operably linked to an influenza virus M DNA, optionally a vector for mRNA production of M1 and M2 is included, wherein if the one or more transcription cassettes for viral segments do not include a transcription cassette comprising a promoter operably linked to an influenza virus NS DNA, optionally a vector for mRNA production of NS1 and NS2 is included, and wherein if the one or more transcription cassettes for vRNA do not include a transcription cassette comprising a promoter operably linked to an influenza virus NA DNA, a vector for mRNA production of NA is included.
9. The set of host cells of claim 1 wherein the non-influenza virus host cell binding protein comprises a filovirus protein, an antibody, an alphavirus protein, a lentivirus protein, a retrovirus protein, albumin, or a rhabdovirus protein.
10. An isolated host cell which produces virus-like particles (VLPs) with at least one and up to eight influenza A or B virus segments and has one or more vectors which include at least one and up to eight transcription cassettes for production of at least one and up to eight viral segments from the genome of influenza A virus selected from:
a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus M DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NS DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NA DNA; or
a transcription cassette comprising a promoter operably linked to an influenza virus HA DNA or a non-influenza host cell binding protein DNA; or
has one or more vectors which include at least one and up to eight transcription cassettes for production of at least one and up to eight viral segments from the genome of influenza B virus selected from:
a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus M DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NS DNA;
a transcription cassette comprising a promoter operably linked to an influenza virus NA and NB DNA; o a transcription cassette comprising a promoter operably linked to an influenza virus HA DNA or a non-influenza host cell binding protein DNA;
and one or more vectors which include transcription cassettes for mRNA production including a transcription cassette comprising a promoter operably linked to an influenza virus PA DNA; a transcription cassette comprising a promoter operably linked to an influenza virus PB1 DNA: a transcription cassette comprising a promoter operably linked to an influenza virus PB2 DNA; a transcription cassette comprising a promoter operably linked to an influenza virus NP DNA; and optionally a transcription cassette comprising a promoter operably linked to an influenza virus HA or a non-influenza host cell binding protein DNA,
wherein if the host cell has a transcription cassette for only one viral segment, the segment encodes a functional influenza virus protein, wherein if the host cell has a transcription cassette for seven viral segments, the host cell does not include sequences corresponding to NA coding or noncoding sequences for production of a NA viral segment and does not include sequence for mRNA production of NA, and wherein if the host cell has a transcription cassette for eight viral segments, at least one of the segments is modified so that a functional influenza virus protein is not expressed from that segment and wherein the modified segment is not the PB2 segment.
11. The host cell of claim 10 which has 1, 2, 3, 4, 5, 6, or 7 of the expression cassettes for production of viral segments.
12. The host cell of claim 10 which stably expresses one or more of the transcription cassettes for mRNA production.
13. The host cell of claim 10 further comprising a transcription cassette comprising a promoter linked to 5′ influenza virus sequences comprising 5′ influenza virus noncoding sequences linked to a DNA of interest linked to Y influenza virus sequences comprising 3′ influenza virus noncoding sequences linked to a transcription termination sequence.