Defective Influenza Virus Particles
The invention relates to the field of influenza virus and the vaccination against flu. The invention provides a conditionally defective influenza virus particle having seven different influenza nucleic acid segments. The invention also provides a conditionally defective influenza virus particle lacking an influenza nucleic acid segment selected from the group of segments essentially encoding acidic polymerase (PA), the basic polymerase 1 (PB1) and the basic polymerase 2 (PB2). In particular, the invention provides defective influenza virus particles having seven different influenza nucleic acid segments and lacking an influenza nucleic acid segment essentially encoding acidic polymerase. Furthermore, the invention provides use of a composition comprising a defective influenza virus particle according to the invention for the production of a pharmaceutical composition directed at generating immunological protection against infection of a subject with an influenza virus, and provides a method for generating immunological protection against infection of a subject with an influenza virus comprising providing a subject in need thereof with a composition comprising such defective influenza virus particle.
1 - 27 . (canceled)
28 . A method for obtaining at least one conditionally defective influenza virus particle comprising:
transfecting a suitable first cell or cells,
with one or more gene constructs derived by internally deleting a nucleic acid encoding an influenza protein whereby said one or more gene constructs are incapable of producing a functional protein, and do not hinder packaging of the gene segments of the virus into viral particles, and
with one or more complementing influenza virus nucleic acid segments encoding an influenza virus, and
with one or more expression plasmids capable of expressing said one or more proteins in said first cell or cells, and
harvesting at least one virus particle from the supernatant of said first cell or cells at a suitable time point after transfection;
transfecting a suitable second cell or cells with one or more expression plasmids capable of expressing said one or more proteins in said second cell or cells;
infecting said second cell or cells with supernatant comprising at least one virus particle obtained from said first cell or cells; and
harvesting at least one virus particle from the supernatant of said second cell or cells at a suitable time point after infection.
29 . A method for obtaining at least one conditionally defective influenza virus particle comprising:
transfecting a suitable cell or cells,
with one or more gene constructs derived by internally deleting a nucleic acid encoding an influenza polymerase whereby said one or more gene constructs are incapable of producing a functional polymerase, and do not hinder packaging of the gene segments of the virus into viral particles, and
with one or more complementing influenza virus nucleic acid segments encoding an influenza virus, and
with one or more expression plasmids capable of expressing said one or more polymerases in said cell, and
harvesting at least one virus particle from the supernatant of said cell or cells at a suitable time point after infection.
30 . The method according to claim 29 , wherein the cell or cells to be infected with supernatant comprising at least one conditionally defective influenza virus particle already express a non-functional polymerase.
31 . The method according to claim 29 , wherein said one or more polymerases comprise one or more of acidic polymerase (PA), basic polymerase 1 (PB1), and basic polymerase 2 (PB2).
32 . The method according to claim 31 , wherein the internal deletion in the PA nucleic acid starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 58 and 207 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 27 and 194 counted from the non-coding region for the PA protein.
33 . The method according to claim 31 , wherein the internal deletion in the PB1 nucleic acid starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 43 and 246 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 24 and 197 counted from the non-coding region for the PB1 protein.
34 . The method according to claim 31 , wherein the internal deletion in the PB2 nucleic acid starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 34 and 234 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 27 and 209 counted from the non-coding region for the PB2 protein.
35 . A method for obtaining at least one conditionally defective influenza virus particle comprising:
transfecting a suitable cell or cells with one or more expression plasmids capable of expressing one or more influenza polymerases in said cell or cells;
infecting said cell or cells with supernatant comprising at least one conditionally defective influenza virus particle; and
harvesting at least one virus particle from the supernatant of said cell or cells at a suitable time point after infection.
36 . The method according to claim 35 , wherein said one or more influenza polymerases comprise one or more of acidic polymerase (PA), basic polymerase 1 (PB1), and basic polymerase 2 (PB2).
37 . A method for obtaining at least one conditionally defective influenza virus particle comprising:
transfecting a suitable first cell or cells,
with one or more gene constructs derived by internally deleting a nucleic acid encoding an influenza polymerase whereby said one or more gene constructs are incapable of producing a functional polymerase, and do not hinder packaging of the gene segments of the virus into viral particles, and
with one or more complementing influenza virus nucleic acid segments encoding an influenza virus, and
with one or more expression plasmids capable of expressing said one or more polymerases in said first cell or cells, and
harvesting at least one virus particle from the supernatant of said first cell or cells at a suitable time point after transfection;
transfecting a suitable second cell or cells with one or more expression plasmids capable of expressing said one or more polymerases in said second cell or cells;
infecting said second cell or cells with supernatant comprising at least one virus particle obtained from said first cell or cells; and
harvesting at least one virus particle from the supernatant of said second cell or cells at a suitable time point after infection.
38 . The method according to claim 37 , wherein the cell or cells to be infected with supernatant comprising at least one conditionally defective influenza virus particle already expresses a non-functional polymerase.
39 . The method according to claim 37 , wherein said one or more polymerases comprise one or more of acidic polymerase (PA), basic polymerase 1 (PB1), and basic polymerase 2 (PB2).
40 . The method according to claim 39 , wherein the internal deletion in the PA nucleic acid starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 58 and 207 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 27 and 194 counted from the non-coding region for the PA protein.
41 . The method according to claim 39 , wherein the internal deletion in the PB1 nucleic acid starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 43 and 246 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 24 and 197 counted from the non-coding region for the PB1 protein.
42 . The method according to claim 39 , wherein the internal deletion in the PB2 nucleic acid starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 34 and 234 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 27 and 209 counted from the non-coding region for the PB2 protein.
43 . An influenza virus particle obtainable by the method according to claim 37 .
44 . An influenza virus particle comprising one or more nucleic acid segments with an internal deletion in at least one segment, said deletion rendering the segment incapable of producing a functional influenza polymerase, and not hindering packaging of the gene segment of the virus into viral particles, wherein the polymerase is chosen from acidic polymerase (PA), basic polymerase 1 (PB1), and basic polymerase 2 (PB2).
45 . An influenza virus particle according to claim 44 , wherein the internal deletion in PA starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 58 and 207 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 27 and 194 counted from the non-coding region for the PA protein.
46 . An influenza virus particle according to claim 44 , wherein the internal deletion in PB1 starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 43 and 246 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 24 and 197 counted from the non-coding region for the PB1 protein.
47 . An influenza virus particle according to claim 44 , wherein the internal deletion in PB2 starts at a 5′-nucleotide situated between, but not encompassing, nucleotides 34 and 234 counted from the non-coding region, and finishes at a 3′-nucleotide situated between, but not encompassing, nucleotides 27 and 209 counted from the non-coding region for the PB2 protein.
48 . A particle according to claim 44 comprising at least one the influenza virus nucleic acid segment encoding at least one viral glycoprotein.
49 . A particle according to claim 45 comprising at least one influenza virus nucleic acid segment encoding the nucleoprotein (NP), the basic polymerase 1 (PB1), the basic polymerase 2 (PB2), the hemagglutinin (HA), the neuraminidase (NA), the matrix proteins (M1 and M2) or the nonstructural proteins (NS1 and NS2).
50 . A particle according to claim 46 comprising at least one influenza virus nucleic acid segment encoding the nucleoprotein (NP), the acid polymerase (PA), the basic polymerase 2 (PB2), the hemagglutinin (HA), the neuraminidase (NA), the matrix proteins (M1 and M2) and the nonstructural protein (NS1 and NS2).
51 . A particle according to claim 47 comprising at least one influenza virus nucleic acid segment encoding the nucleoprotein (NP), the acid polymerase (PA), the basic polymerase 1 (PB1), the hemagglutinin (HA), the neuraminidase (NA), the matrix proteins (M1 and M2) and the nonstructural protein (NS1 and NS2).
52 . An influenza virus particle according to claim 44 , comprising at least one influenza virus nucleic acid segment that is derived from influenza A virus.
53 . An influenza virus particle according to claim 44 , comprising a nucleic acid not encoding an influenza peptide.
54 . A composition comprising an influenza virus particle according to claim 44 .
55 . A cell comprising an influenza virus particle according to claim 44 .
56 . A cell according to claim 55 , comprising one or more influenza virus polymerases wherein the polymerase is chosen from acidic polymerase (PA), basic polymerase 1 (PB1), and basic polymerase 2 (PB2).
57 . A composition comprising a cell or material derived from a cell according to claim 55 .
58 . A method for generating immunological protection against infection of a subject with an influenza virus, comprising administering to a subject in need thereof a composition according to claim 54 .
59 . A method for generating immunological protection against infection of a subject with an influenza virus, comprising administering to a subject in need thereof a composition according to claim 57 .
60 . A method for delivery of a nucleic acid not encoding an influenza peptide to a cell, comprising providing said cell with a defective influenza virus particle according to claim 53 .
61 . A method for delivery of a nucleic acid not encoding an influenza peptide to a subject, comprising providing said subject with a defective influenza virus particle according to claim 53 .