Attenuated influenza vaccines and uses thereof

Abstract

Provided herein are attenuated influenza viruses and methods of making attenuated influenza viruses.

What is claimed is: 
     
         1 . A modified influenza A virus comprising a PB1 polymerase having one or more mutations in amino acids 310 to 325. 
     
     
         2 . The virus of  claim 1 , wherein the PB1 polymerase comprises a leucine to glutamine substitution at position 319 (L319Q). 
     
     
         3 . The virus of  claim 1 , wherein the PB1 polymerase further comprises one or more mutations selected from the group consisting of a lysine to glutamic acid substitution at position 391 (K391E), a glutamic acid to glycine substitution at position 581 (E581G) and an alanine to threonine substitution at position 661 (A661T). 
     
     
         4 . The virus of  claim 1 , wherein the virus further comprises a PB2 polymerase comprising an asparagine to serine substitution at position 265 (N265S). 
     
     
         5 . The virus of  claim 1 , wherein the virus further comprises an influenza virus nucleoprotein (NP) comprising an aspartic acid to glycine substitution at position 35 (D34G). 
     
     
         6 . The virus of  claim 1 , wherein the influenza A virus is selected from the group consisting of an H2N2 virus, an H3N2 virus, an H1N1 virus, an H9N2 virus and an H5N1 virus. 
     
     
         7 . The virus of  claim 1 , wherein the influenza A virus is A/Ann Arbor/6/60 (H2N2). 
     
     
         8 . The virus of  claim 1 , wherein the virus is a live attenuated influenza A virus with reduced growth from about 37° C. to about 39° C., as compared to an influenza A virus comprising a PB1 polymerase lacking one or more mutations in amino acids 310 to 325. 
     
     
         9 . An immunogenic composition comprising the virus of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         10 . A method for eliciting an immune response against an influenza virus in a subject, comprising administering an effective dose of the immunogenic composition of  claim 9  to the subject. 
     
     
         11 . A method of treating or reducing the risk of an influenza infection in a subject, comprising administering to a subject with an influenza infection or susceptible to an influenza infection an effective dose of the immunogenic composition of  claim 9 . 
     
     
         12 . A recombinant nucleic acid encoding a PB1 polymerase of an influenza A virus, wherein the nucleic acid encodes a PB1 polymerase having one or more mutations in amino acids 310 to 325. 
     
     
         13 . The nucleic acid of  claim 12 , wherein the nucleic acid encodes a PB1 polymerase comprising a leucine to glutamine substitution at position 319 (L319Q). 
     
     
         14 . The nucleic acid of  claim 12 , wherein the nucleic acid encodes a PB1 polymerase comprising a leucine to glutamine substitution at position 319 (L319Q) and one or mutations selected from the group consisting of a lysine to glutamic acid substitution at position 391 (K391E), a glutamic acid to glycine substitution at position 581 (E581G) and an alanine to threonine substitution at position 661 (A661T). 
     
     
         15 . A vector comprising the nucleic acid of  claim 12 . 
     
     
         16 . A method of producing the influenza virus of  claim 1 , comprising:
 (a) transfecting a population of host cells with one or more vectors comprising
 (i) nucleic acid sequences encoding the internal genome segments of an influenza A virus and; 
 (ii) a nucleic acid encoding a PB1 polymerase with one or more mutations in amino acids 310 to 325; 
   (b) culturing the host cells; and   (c) recovering the modified influenza A virus.   
     
     
         17 . The method of  claim 16 , wherein the nucleic acid encoding the PB1 polymerase encodes a PB1 polymerase comprising a leucine to glutamine substitution at position 319 (L319Q). 
     
     
         18 . The method of  claim 16 , wherein the nucleic acid encoding the PB1 polymerase encodes a PB1 polymerase comprising L319Q and one or mutations selected from the group consisting of a lysine to glutamic acid substitution at position 391 (K391E), a glutamic acid to glycine substitution at position 581 (E581G) and an alanine to threonine substitution at position 661 (A661T). 
     
     
         19 . The method of  claim 16 , wherein the one or more vectors further comprise a nucleic acid encoding a PB2 polymerase comprising a N265S mutation. 
     
     
         20 . The method of  claim 16 , wherein the one or more vectors comprise a nucleic acid encoding an NP protein comprising a D34G mutation. 
     
     
         21 . The method of  claim 16 , wherein the influenza A virus is selected from the group consisting of an H2N2 virus, an H3N2 virus, an H1N1 virus, an H9N2 virus and an H5N1 virus. 
     
     
         22 . The method of  claim 16 , wherein the cells are Vero cells, MDCK cells or CEK cells. 
     
     
         23 . A method for producing an influenza immunogen comprising:
 (a) infecting a population of cells with the virus of  claim 1 ;   (b) culturing the cells;   (c) harvesting the virus from the culture of step b); and   (d) preparing an immunogen with the harvested virus.   
     
     
         24 . The method of  claim 23 , wherein the cells are mammalian cells or avian cells. 
     
     
         25 . A population of cells comprising the virus of  claim 1 . 
     
     
         26 . A modified influenza A virus comprising a PB1 polymerase with one or more mutations in amino acids 310 to 325, wherein at least one of the mutations is selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at position 319 (L319E/D/N) and a substitution at position 323. 
     
     
         27 . The virus of  claim 26 , wherein the PB1 polymerase comprises a leucine to glutamic acid substitution at position 319 (L319E). 
     
     
         28 . The virus of  claim 26 , wherein the PB1 polymerase comprises a leucine to asparagine substitution at position 319 (L319N). 
     
     
         29 . The virus of  claim 26 , wherein the PB1 polymerase comprises a threonine to glutamic acid, aspartic acid, glutamine, asparagine or alanine substitution at position 323. 
     
     
         30 . The virus of  claim 26 , wherein the PB1 polymerase further comprises one or more mutations selected from the group consisting of a lysine to glutamic acid substitution at position 391 (K391E), a glutamic acid to glycine substitution at position 581 (E581G) and an alanine to threonine substitution at position 661 (A661T). 
     
     
         31 . The virus of  claim 26 , wherein the virus further comprises a PB2 polymerase comprising an asparagine to serine substitution at position 265 (N265S). 
     
     
         32 . The virus of  claim 26 , wherein the virus further comprises an influenza virus nucleoprotein (NP) comprising an aspartic acid to glycine substitution at position 34 (D34G). 
     
     
         33 . The virus of  claim 26 , wherein the influenza A virus is selected from the group consisting of an H2N2 virus, an H3N2 virus, an H1N1 virus, an H9N2 virus and an H5N1 virus. 
     
     
         34 . The virus of  claim 26 , wherein the influenza A virus is A/Ann Arbor/6/60 (H2N2) or (A/Puerto Rico/8/34/H1 N1)(PR8). 
     
     
         35 . The virus of  claim 26 , wherein the virus is a live attenuated influenza A virus with reduced growth from about 37° C. to about 39° C., as compared to an influenza A virus comprising a PB1 polymerase lacking one or more mutations in amino acids 310 to 325. 
     
     
         36 . An immunogenic composition comprising the virus of  claim 26  and a pharmaceutically acceptable carrier. 
     
     
         37 . A method for eliciting an immune response against an influenza virus in a subject, comprising administering an effective dose of the immunogenic composition of  claim 36  to the subject. 
     
     
         38 . A method of treating or reducing the risk of an influenza infection in a subject, comprising administering to a subject with an influenza infection or at risk of exposure to an influenza infection an effective dose of the immunogenic composition of  claim 36 . 
     
     
         39 . A recombinant nucleic acid encoding a PB1 polymerase of an influenza A virus, wherein the nucleic acid encodes a PB1 polymerase having one or more mutations in amino acids 310 to 325 and wherein at least one of the mutations is selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at position 319 (L319E/D/N), and a substitution at position 323. 
     
     
         40 . The recombinant nucleic acid of  claim 39 , wherein the PB1 polymerase comprises a L319E mutation. 
     
     
         41 . The recombinant nucleic acid of  claim 39 , wherein the PB1 polymerase comprises a L319N mutation. 
     
     
         42 . The nucleic acid of  claim 39 , wherein the nucleic acid encodes a PB1 polymerase having one or more mutations selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at position 319 (L319E/D/N) and a substitution at position 323; and one or mutations selected from the group consisting of a lysine to glutamic acid substitution at position 391 (K391E), a glutamic acid to glycine substitution at position 581 (E581G) and an alanine to threonine substitution at position 661 (A661T). 
     
     
         43 . A vector comprising the nucleic acid of  claim 39 . 
     
     
         44 . A method of producing the influenza virus of  claim 26 , comprising:
 (a) transfecting a population of host cells with one or more vectors comprising   (i) nucleic acid sequences encoding the internal genome segments of an influenza A virus and;   (ii) a nucleic acid encoding a PB1 polymerase of an influenza A virus having one or more mutations in amino acids 310 to 325, wherein at least one of the mutations is selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at position 319 (L319E/D/N) and a substitution at position 323;   (b) culturing the host cells; and   (c) recovering the influenza A virus.   
     
     
         45 . The method of  claim 44 , wherein the nucleic acid encodes a PB1 polymerase having one or more mutations selected from the group consisting of a leucine to glutamic acid, aspartic acid or asparagine substitution at position 319 (L319E/D/N), a substitution at position 323 and one or mutations selected from the group consisting of a lysine to glutamic acid substitution at position 391 (K391E), a glutamic acid to glycine substitution at position 581 (E581G) and an alanine to threonine substitution at position 661 (A661T). 
     
     
         46 . The method of  claim 44 , wherein the one or more vectors further comprise a nucleic acid encoding a PB2 polymerase comprising a N265S mutation. 
     
     
         47 . The method of  claim 44 , wherein the one or more vectors further comprise a nucleic acid encoding an NP protein comprising a D34G mutation. 
     
     
         48 . The method of  claim 44 , wherein the influenza A virus is selected from the group consisting of an H2N2 virus, an H3N2 virus, an H1N1 virus, an H9N2 virus and an H5N1 virus. 
     
     
         49 . The method of  claim 44 , wherein the cells are Vero cells, MDCK cells or CEK cells. 
     
     
         50 . A method for producing an influenza immunogen comprising:
 (a) infecting a population of cells with the virus of  claim 26 ;   (b) culturing the cells;   (c) harvesting the virus from the culture of step b); and   (d) preparing an immunogen with the harvested virus.   
     
     
         51 . The method of  claim 51 , wherein the cells are mammalian cells or avian cells. 
     
     
         52 . A population of cells comprising the virus of  claim 26 .