Influenza virus reassortment

Abstract

New influenza donor strains for the production of reassortant influenza A viruses are provided.

1 - 24 . (canceled) 
     
     
         25 . A reassortant influenza A virus comprising an HA segment, an NA segment and backbone segments PA, PB1, PB2, NP, NS and M, wherein the backbone segments are from two or more donor strains, wherein (a) the HA segment and the PB1 segment are from different influenza A strains with the same influenza virus HA subtype, (b) wherein the HA segment and the PB1 segment are from different influenza A strains with different influenza virus HA subtypes, wherein the PB1 segment is not from an influenza virus with a H3 HA subtype and/or wherein the HA segment is not from an influenza virus with a H1 or H5 HA subtype, or (c) at least one backbone segment is from the A/California/07/09 influenza strain. 
     
     
         26 . The reassortant influenza A virus of  claim 25 , wherein the HA segment and the PB1 segment are from a H1 influenza strain. 
     
     
         27 . The reassortant influenza A virus of  claim 25 , wherein the reassortant influenza A virus comprises (b) and the PB1 segment is from a H1 virus and/or wherein the HA segment is from a H3 influenza vims. 
     
     
         28 . The reassortant influenza A virus of  claim 25 , wherein the reassortant influenza A virus comprises (c) and the at least one backbone segment is the PB1 segment. 
     
     
         29 . The reassortant influenza A virus of  claim 28 , wherein the PB1 segment has at least 95%, at least 99% identity, or 100% identity with the sequence of SEQ ID NO: 16. 
     
     
         30 . The reassortant influenza A virus of  claim 25 , wherein the reassortant influenza A virus comprises (c) and the HA segment is from a H1 influenza strain. 
     
     
         31 . The reassortant influenza A virus of  claim 25 , wherein the PB1 segment and the PB2 segment are from the same donor strain. 
     
     
         32 . The reassortant influenza A virus of  claim 25 , wherein the segments are selected from the group consisting of:
 a) the PA segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 1;   b) the PB2 segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 3;   c) the M segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 5;   d) the NP segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 4; and/or   e) the NS segment having at least 95% or 99% identity to the sequence of SEQ ID NO: 6.   
     
     
         33 . The reassortant influenza A virus of  claim 32 , wherein the PA segment has 95% identity to the sequence of SEQ ID NO: 1, the PB2 segment has 95% identity to the sequence of SEQ ID NO: 3, the M segment has 95% identity to the sequence of SEQ ID NO: 5, the NP segment has 95% identity to the sequence of SEQ ID NO: 4 and the NS segment has 95% identity to the sequence of SEQ ID NO: 6. 
     
     
         34 . The reassortant influenza A virus of  claim 33 , wherein the PA segment has the sequence of SEQ ID NO: 1, the PB2 segment has the sequence of SEQ ID NO: 3, the M segment has the sequence of SEQ ID NO: 5, the NP segment has the sequence of SEQ ID NO: 4 and the NS segment has the sequence of SEQ ID NO: 6. 
     
     
         35 . The reassortant influenza A virus of  claim 25  comprising backbone segments (i) from two, three or four donor strains, wherein each donor strain provides more than one backbone segment, (ii) from two or more donor strains, wherein the PB1 segment is not from the A/Texas/1/77 influenza strain, or (iii) from two or more donor strains, wherein at least the PA, NP, or M segment are not from A/Puerto Rico/8/34. 
     
     
         36 . The reassortant influenza A virus of  claim 25 , wherein at least one of the backbone segments is selected from the group consisting of:
 a) the PB2 segment which has lysine in the position corresponding to amino acid 389 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm; and/or   b) the PB2 segment which has asparagine in the position corresponding to amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm; and/or   c) the PA segment which has lysine in the position corresponding to amino acid 327 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm; and/or   d) the PA segment which has aspartic acid in the position corresponding to amino acid 444 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm; and/or   e) the PA segment which has aspartic acid in the position corresponding to amino acid 675 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm; and/or   f) the NP segment which has threonine in the position corresponding to amino acid 27 of SEQ ID NO: 4 when aligned to SEQ ID NO: 4 using a pairwise alignment algorithm; and/or   g) the NP segment which has asparagine in the position corresponding to amino acid 375 of SEQ ID NO: 4 when aligned to SEQ ID NO: 4, using a pairwise alignment algorithm.   
     
     
         37 . The reassortant influenza A strain of  claim 36 , wherein (i) the PB2 segment has lysine in the position corresponding to amino acid 389 of SEQ ID NO: 3 and asparagine in the position corresponding to amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm, (ii) the PA segment has lysine in the position corresponding to amino acid 327: aspartic acid in the position corresponding to amino acid 444 of SEQ ID NO: 1 and aspartic acid in the position corresponding to amino acid 675 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm, or (iii) the NP genome segment has threonine in the position corresponding to amino acid 27 of SEQ ID NO: 4 and asparagine in the position corresponding to amino acid 375 when aligned to SEQ ID NO: 4. using a pairwise alignment algorithm, or (iv) the influenza A strain is a H1 strain. 
     
     
         38 . The reassortant influenza A strain of  claim 25 , wherein the PB2 segment has lysine in the position corresponding to amino acid 389 of SEQ ID NO: 3 and asparagine in the position corresponding to amino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignment algorithm, PA genome segment has lysine in the position corresponding to amino acid 327; aspartic acid in the position corresponding to amino acid 444 of SEQ ID NO: 1 and aspartic acid in the position corresponding to amino acid 675 when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm, and an NP genome segment has threonine in the position corresponding to amino acid 27 of SEQ ID NO: 4 and asparagine in the position corresponding to amino acid 375 when aligned to SEQ ID NO: 4, using a pairwise alignment algorithm 
     
     
         39 . A method of preparing a reassortant influenza A virus of  claim 25  comprising steps of
 (i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza A virus wherein the expression construct(s) encode the backbone segments from two or more donor strains and wherein the HA and PB1 genome segments are from different influenza strains which have the same influenza HA subtype; and 
 (ii) culturing the culture host in order to produce the reassortant influenza A virus of  claim 25 , wherein the reassortant influenza A virus comprises (a). 
 
     
     
         40 . The method of  claim 39 , wherein (1) the expression construct(s) do/does not encode the PB1 segment from the A/Texas/1/77 influenza strain, (2) the at least one expression construct comprises a sequence having at least 90% or 100% identity with the sequence of SEQ ID NO: 22, (3) the expression construct(s) further comprise(s) one or more of the sequences having at least 90% identity or 100% identity with the sequences of SEQ ID Nos.: 9 and/or 11 to 14, or (4) further comprising the step (iii) of purifying the reassortant virus obtained in step (ii). 
     
     
         41 . A method of preparing a reassortant influenza A virus of  claim 28  comprising steps of
 (i) introducing into a culture host one or more expression construct(s) which encode(s) the viral segments required to produce an influenza A virus wherein the expression construct(s) encode the backbone segments from two or more donor strains and wherein the PB1 backbone viral segment is from A/California/07/09; and 
 (ii) culturing the culture host in order to produce the reassortant influenza A virus of  claim 28 . 
 
     
     
         42 . The method of  claim 39 , wherein the expression construct(s) are (3) and comprise(s) all of the sequences having at least 90% identity or 100% identity with the sequences of SEQ ID Nos.: 9 and 11 to 14. 
     
     
         43 . The method of  claim 40 , wherein the HA segment is from a H1 influenza virus. 
     
     
         44 . A method for producing influenza viruses comprising steps of (a) infecting a culture host with the reassortant influenza virus of  claim 25 ; (b) culturing the host from step (a) to produce the virus; and optionally (c) purifying the virus produced in step (b). 
     
     
         45 . A method of preparing a vaccine, comprising the steps of (a) preparing a virus by the method of  claim 44  and (b) preparing a vaccine from the virus. 
     
     
         46 . The method of  claim 44 , wherein the culture host is an embryonated hen egg. 
     
     
         47 . The method of  claim 44 , wherein the culture host is a mammalian cell, optionally an MDCK (such as MDCK 33016 (DSM ACC2219)), Vero or PerC6 cell. 
     
     
         48 . The method of  claim 47 , wherein the cell grows adherently or in suspension. 
     
     
         49 . The method of  claim 45 , wherein step (b) involves inactivating the virus. 
     
     
         50 . The method of  claim 45 , wherein the vaccine is a whole virion vaccine, a split virion vaccine. a surface antigen vaccine, or a virosomal vaccine. 
     
     
         51 . The method of  claim 45 , wherein the vaccine contains less than 10 ng of residual host cell DNA per dose. 
     
     
         52 . The method of  claim 45 , wherein at least one of the influenza strains is of the H1, H2, H5, H7 or H9 subtype. 
     
     
         53 . An expression system comprising one or more expression construct(s) comprising the vRNA encoding segments of an influenza A virus wherein the expression construct(s) encode(s) the backbone viral segments from two or more influenza donor strains, wherein (i) the HA and PB1 segments are from two different influenza strains with the same influenza HA subtype, (ii) the HA and PB1 segments are from two different influenza strains with different influenza virus HA subtypes, wherein the expression construct(s) do(es) not encode the PB1 segment from an influenza virus with a H3 HA subtype and/or wherein the expression construct(s) do(es) not encode the HA segment from an influenza virus with a H1 or H5 HA subtype. or (iii) wherein the PB1 segment is from A/California/07/09. 
     
     
         54 . The expression system of  claim 53 , wherein (a) the expression construct(s) may further comprise the vRNAs which encode the PB2, NP, NS, M and PA segments from PR8-X,(b) wherein the at least one expression construct comprises a sequence having at least 90%, at least 95%, at least 99% or 100% identity with the sequence of SEQ ID NO: 22, (c) the expression construct(s) further comprise(s) one or more of the sequences having at least 90%, at least 95%, at least 99% or 100% identity with the sequences of SEQ ID Nos.: 9, and/or 11 to 14. or (d) the expression construct(s) comprise(s) all of the sequences having at least 90%, at least 95%, at least 99% or 100% identity with the sequences of SEQ ID Nos.: 9 and 11 to 14. 
     
     
         55 . A host cell comprising the expression system of  claim 53 . 
     
     
         56 . The host cell of  claim 55 , wherein the host cell is a mammalian cell, optionally an MDCK, Vero or PerC6 cell.