Recombinant influenza viruses with stabilized HA for replication in eggs
Modified influenza virus neuraminidases are described herein that improve viral replication, thus improving the yield of vaccine viruses. Expression of such modified neuraminidases by influenza virus may also stabilize co-expressed hemagglutinins so that the hemagglutinins do not undergo mutation or decrease the need for HA binding to cells.
1. An isolated recombinant influenza virus comprising a selected neuraminidase (NA) viral segment encoding a plurality of selected residues in NA,
wherein the selected NA viral segment encodes a NA having an isoleucine (I) at residue 148, and wherein the plurality of selected residues in the NA includes when the NA does not encode an aspartic acid (D) at position 151, does not encode an asparagine (N) at position 245, does not encode a threonine (T) at position 329, does not encode a lysine (K) at position 344, does not encode a glycine (G) at position 346, does not encode a histidine (H) at residue 347, or does not encode a threonine at position 369, or any combination thereof, wherein the numbering is based on N2, wherein the recombinant influenza virus has enhanced replication in avian eggs, has reduced sialidase activity, enhanced binding to a2-3 sialosides, or has a reduction in hemagglutinin (HA) mutations when grown in avian eggs relative to a corresponding influenza virus that has a NA that encodes a threonine or lysine at residue 148, encodes an aspartic acid at residue 151, encodes an asparagine at residue 245, encodes a threonine at residue 329, encodes a lysine at residue 344, encodes a glycine at residue 346, encodes a histidine at residue 347, and encodes a threonine at position 369, wherein the numbering is based on N2 of SEQ ID NO:53.
2. The isolated recombinant influenza virus of claim 1 wherein the selected NA segment encodes glutamic acid (E) at residue 151, serine (S), threonine, glycine, alanine (A), leucine or isoleucine at residue 245, serine, glycine, alanine, leucine or isoleucine residue 329, glutamic acid, aspartic acid or histidine at residue 344, valine, leucine, isoleucine, threonine or serine at reside 346, glycine, alanine, valine, leucine, isoleucine or threonine at residue 347, or lysine, histidine, aspartic acid or glutamic acid at residue 369.
3. The isolated recombinant influenza virus of claim 1 wherein the residue at position 151 is E, N or Q or position 344 is E, D or H.
4. The isolated recombinant influenza virus of claim 1 wherein the residue at position 245 is S, T, I, L, A, V, or G.
5. The isolated recombinant influenza virus of claim 1 wherein the residue at position 347 is G, Q, S, T, Y, C or W.
6. The isolated recombinant influenza virus of claim 1 wherein the residue at position 369 is K, H, R, E, P, or D.
7. The isolated recombinant influenza virus of claim 1 wherein the residue at position 329 is serine, valine, alanine, glycine, isoleucine or leucine.
8. The isolated recombinant influenza virus of claim 1 wherein the residue at position 346 is V, S, T, A, I, or L.
9. The isolated recombinant influenza virus of claim 1 wherein the residue at position 151 is E, N or Q, the residue at position 245 is S, T, I, L, A, W, Y, P, V, or G, the residue at position 329 is S, I, L, A, W, Y, P, V, or G, the residue at position 344 is E, H, D, N or Q, the residue at position 346 is V, S, T, I, L, A, W, Y, or P, the residue at position 347 is G, Q, S, T, Y, C or W, or the residue at position 369 is K, H, R, E, P, or D.
10. The isolated recombinant influenza virus of claim 1 which is a reassortant.
11. The isolated recombinant influenza virus of claim 1 wherein the NA viral segment encodes a NA that has at least 90% amino acid sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74, or has at least 90% amino acid sequence identity to a NA encoded by any one of SEQ ID Nos. 55-59.
12. The isolated recombinant influenza virus of claim 1 wherein the NA viral segment encodes a N2, N3, N7, or N9 or wherein HA is H1, H2, H3, H5, H7, or H9.
13. The isolated recombinant influenza virus of claim 1 wherein polymerase A (PA), polymerase B1 (PB1), polymerase B2 (PB2), nucleoprotein (NP), matrix (M), and non-structural (NS) viral segments have at least 85% nucleic acid sequence identity to SEQ ID Nos. 24 to 29 or 39 to 44 or encode a polypeptide having at least 80% amino acid sequence identity to a polypeptide encoded by SEQ ID Nos. 24 to 29 or 39 to 44.
14. The isolated recombinant influenza virus of claim 1 wherein PB2 has I, A, L, or G at residue 147 based on the numbering of a corresponding polypeptide encoded by SEQ ID NO:26.
15. The isolated recombinant influenza virus of claim 1 which has one or more of PB2-I504V, PB1-M40L/G180W, PA-R401K, NP-I116L, and NS1-A30P/R118K based on the numbering of a corresponding polypeptide encoded by one of SEQ ID Nos. 24-27 or 29.
16. A method of immunizing an avian or a mammal, comprising: administering to the avian or the mammal a composition having an effective amount of the virus of claim 1 .
17. The isolated recombinant influenza virus of claim 1 wherein the residue at position 245 is S, the residue at position 346 is V, the residues at position 347 is G, or the residue at position 369 is K.