Clin Exp Vaccine Res.  2012 Jul;1(1):35-49. 10.7774/cevr.2012.1.1.35.

Principles underlying rational design of live attenuated influenza vaccines

• Jang YH
• Seong BL

• KMID: 2278787
• DOI: http://doi.org/10.7774/cevr.2012.1.1.35

Abstract

Despite recent innovative advances in molecular virology and the developments of vaccines, influenza virus remains a serious burden for human health. Vaccination has been considered a primary countermeasure for prevention of influenza infection. Live attenuated influenza vaccines (LAIVs) are particularly attracting attention as an effective strategy due to several advantages over inactivated vaccines. Cold-adaptation, as a classical means for attenuating viral virulence, has been successfully used for generating safe and effective donor strains of LAIVs against seasonal epidemics and occasional pandemics. Recently, the advent of reverse genetics technique expedited a variety of rational strategies to broaden the pool of LAIVs. Considering the breadth of antigenic diversity of influenza virus, the pool of LAIVs is likely to equip us with better options for controlling influenza pandemics. With a brief reflection on classical attenuating strategies used at the initial stage of development of LAIVs, especially on the principles underlying the development of cold-adapted LAIVs, we further discuss and outline other attenuation strategies especially with respect to the rationales for attenuation, and their practicality for mass production. Finally, we propose important considerations for a rational vaccine design, which will provide us with practical guidelines for improving the safety and effectiveness of LAIVs.

Keyword

Influenza live attenuated vaccine; Cold-adaptation; Attenuation strategy; Cross protection

MeSH Terms

Antigenic Variation
Cross Protection
Humans
Influenza Vaccines
Influenza, Human
Orthomyxoviridae
Pandemics
Reverse Genetics
Seasons
Tissue Donors
Vaccination
Vaccines, Inactivated
Influenza Vaccines
Vaccines, Inactivated

Figure

• 

  Fig. 1 Generation of reassortant live attenuated influenza vaccine (LAIV). LAIVs are made by genetic reassortment between attenuated donor strain and virulent circulating virus. Live vaccine virus consists of six internal genes derived from the attenuated donor strain and the surface genes, change to hematoglutinin and neuraminidase, from the virulent circulating virus. As a result, the vaccine demonstrates attenuated phenotype and carries desired antigen proteins.

• 

  Fig. 2 Reverse genetics approach for generating live attenuated influenza vaccines (LAIVs). Each of six internal genes of attenuated donor strain and two surface genes of circulating wild type virus is inserted into bidirectional expression and transcription plasmid. The eight plasmids are co-transfected into 293T cells, where viral RNAs and proteins are produced, ultimately resulting in the formation of desired vaccine virus particle. PB, polymerase basic; PA, polymerase acidic; HA, hematoglutinin NP, nucleoprotein; M, matrix protein; NS, nonstructural protein 1; NA, neuraminidase.

• 

  Fig. 3 Rational design of live attenuated vaccine. As the safety level of a live vaccine increases, the efficacy of the vaccine is likely to decrease due to the inefficient replication of the vaccine in host (A). Further, the attenuation of viral virulence is often achieved at the expanse of productivity of the vaccine because of the simultaneous loss of viral viability in production hosts (B). A rational vaccine design therefore should carefully consider these two aspects for the vaccine to be potentially practical for clinical use.

Reference

1. Lamb RA, Krug RM. Knipe DM, Howley PM, Griffin DE, editors. Orthomyxoviridae: the viruses and their replication. Fields virology. 2001. 4th ed. Philadelphia: Lippincott Williams & Wilkins;1487–1532.

2. Fouchier RA, Munster V, Wallensten A, et al. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J Virol. 2005. 79:2814–2822.

3. Webster RG, Peiris M, Chen H, Guan Y. H5N1 outbreaks and enzootic influenza. Emerg Infect Dis. 2006. 12:3–8.

4. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev. 1992. 56:152–179.

5. Taubenberger JK, Morens DM. 1918 Influenza: the mother of all pandemics. Emerg Infect Dis. 2006. 12:15–22.

6. Park AW, Glass K. Dynamic patterns of avian and human influenza in east and southeast Asia. Lancet Infect Dis. 2007. 7:543–548.

7. de Jong MD, Hien TT. Avian influenza A (H5N1). J Clin Virol. 2006. 35:2–13.

8. Fouchier RA, Garcia-Sastre A, Kawaoka Y. Pause on avian flu transmission studies. Nature. 2012. 481:443.

9. Cohen J. Avian influenza: surprising twist in debate over lab-made H5N1. Science. 2012. 335:1155–1156.

10. Fiore AE, Bridges CB, Cox NJ. Seasonal influenza vaccines. Curr Top Microbiol Immunol. 2009. 333:43–82.

11. Suguitan AL Jr, McAuliffe J, Mills KL, et al. Live, attenuated influenza A H5N1 candidate vaccines provide broad cross-protection in mice and ferrets. PLoS Med. 2006. 3:e360.

12. Fan S, Gao Y, Shinya K, et al. Immunogenicity and protective efficacy of a live attenuated H5N1 vaccine in nonhuman primates. PLoS Pathog. 2009. 5:e1000409.

13. Chen GL, Min JY, Lamirande EW, et al. Comparison of a live attenuated 2009 H1N1 vaccine with seasonal influenza vaccines against 2009 pandemic H1N1 virus infection in mice and ferrets. J Infect Dis. 2011. 203:930–936.

14. Yang P, Duan Y, Wang C, et al. Immunogenicity and protective efficacy of a live attenuated vaccine against the 2009 pandemic A H1N1 in mice and ferrets. Vaccine. 2011. 29:698–705.

15. Beare AS, Bynoe ML. Attenuation of human influenza A viruses. Br Med J. 1969. 4:198–201.

16. Morris CA, Freestone DS, Stealey VM, Oliver PR. Recombinant WRL 105 strain live attenuated influenza vaccine: immunogenicity, reactivity, and transmissibility. Lancet. 1975. 2:196–199.

17. McCahon D, Beare AS, Stealey V. The production of live attenuated influenza A strains by recombination with A/Okuda/57 (H2N2). Postgrad Med J. 1976. 52:389–394.

18. Beare AS, Schild GC, Craig JW. Trials in man with live recombinants made from A/PR/8/34 (H0 N1) and wild H3 N2 influenza viruses. Lancet. 1975. 2:729–732.

19. Clements ML, Snyder MH, Buckler-White AJ, Tierney EL, London WT, Murphy BR. Evaluation of avian-human reassortant influenza A/Washington/897/80 x A/Pintail/119/79 virus in monkeys and adult volunteers. J Clin Microbiol. 1986. 24:47–51.

20. Murphy BR, Buckler-White AJ, London WT, et al. Avian-human reassortant influenza A viruses derived by mating avian and human influenza A viruses. J Infect Dis. 1984. 150:841–850.

21. Sears SD, Clements ML, Betts RF, Maassab HF, Murphy BR, Snyder MH. Comparison of live, attenuated H1N1 and H3N2 cold-adapted and avian-human influenza A reassortant viruses and inactivated virus vaccine in adults. J Infect Dis. 1988. 158:1209–1219.

22. Snyder MH, Buckler-White AJ, London WT, Tierney EL, Murphy BR. The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys. J Virol. 1987. 61:2857–2863.

23. Treanor JJ, Tierney EL, London WT, Murphy BR. Characterization of the attenuating M and NP gene segments of the avian influenza A/Mallard/78 virus during in vitro production of avian-human reassortant vaccine viruses and after replication in humans and primates. Vaccine. 1991. 9:495–501.

24. Clements ML, Subbarao EK, Fries LF, Karron RA, London WT, Murphy BR. Use of single-gene reassortant viruses to study the role of avian influenza A virus genes in attenuation of wild-type human influenza A virus for squirrel monkeys and adult human volunteers. J Clin Microbiol. 1992. 30:655–662.

25. Subbarao K, Webster RG, Kawaoka Y, Murphy BR. Are there alternative avian influenza viruses for generation of stable attenuated avian-human influenza A reassortant viruses? Virus Res. 1995. 39:105–118.

26. Maassab HF, DeBorde DC. Development and characterization of cold-adapted viruses for use as live virus vaccines. Vaccine. 1985. 3:355–369.

27. Ada GL, Jones PD. The immune response to influenza infection. Curr Top Microbiol Immunol. 1986. 128:1–54.

28. Klimov AI, Egorov AY, Gushchina MI, et al. Genetic stability of cold-adapted A/Leningrad/134/47/57 (H2N2) influenza virus: sequence analysis of live cold-adapted reassortant vaccine strains before and after replication in children. J Gen Virol. 1995. 76(Pt 6):1521–1525.

29. Smorodincev AA. The efficacy of live influenza vaccines. Bull World Health Organ. 1969. 41:585–588.

30. Maassab HF. Adaptation and growth characteristics of influenza virus at 25 degrees c. Nature. 1967. 213:612–614.

31. Maassab HF. Biologic and immunologic characteristics of cold-adapted influenza virus. J Immunol. 1969. 102:728–732.

32. Alexandrova GI, Polezhaev FI, Budilovsky GN, et al. Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees. Infect Immun. 1984. 44:734–739.

33. Alexandrova GI, Smorodintsev AA. Obtaining of an additionally attenuated vaccinating cryophil influenza strain. Rev Roum Inframicrobiol. 1965. 2:179–186.

34. Kendal AP, Maassab HF, Alexandrova GI, Ghendon YZ. Development of cold-adapted recombinant live, attenuated influenza A vaccines in the U.S.A. and U.S.S.R. Antiviral Res. 1982. 1:339–365.

35. Ghendon YZ, Polezhaev FI, Lisovskaya KV, Medvedeva TE, Alexandrova GI, Klimov AI. Recombinant cold-adapted attenuated influenza A vaccines for use in children: molecular genetic analysis of the cold-adapted donor and recombinants. Infect Immun. 1984. 44:730–733.

36. Klimov AI, Cox NJ, Yotov WV, Rocha E, Alexandrova GI, Kendal AP. Sequence changes in the live attenuated, cold-adapted variants of influenza A/Leningrad/134/57 (H2N2) virus. Virology. 1992. 186:795–797.

37. Herlocher ML, Maassab HF, Webster RG. Molecular and biological changes in the cold-adapted "master strain" A/AA/6/60 (H2N2) influenza virus. Proc Natl Acad Sci U S A. 1993. 90:6032–6036.

38. Herlocher ML, Clavo AC, Maassab HF. Sequence comparisons of A/AA/6/60 influenza viruses: mutations which may contribute to attenuation. Virus Res. 1996. 42:11–25.

39. Murphy BR, Coelingh K. Principles underlying the development and use of live attenuated cold-adapted influenza A and B virus vaccines. Viral Immunol. 2002. 15:295–323.

40. Snyder MH, Betts RF, DeBorde D, et al. Four viral genes independently contribute to attenuation of live influenza A/Ann Arbor/6/60 (H2N2) cold-adapted reassortant virus vaccines. J Virol. 1988. 62:488–495.

41. Subbarao EK, Perkins M, Treanor JJ, Murphy BR. The attenuation phenotype conferred by the M gene of the influenza A/Ann Arbor/6/60 cold-adapted virus (H2N2) on the A/Korea/82 (H3N2) reassortant virus results from a gene constellation effect. Virus Res. 1992. 25:37–50.

42. Snyder MH, Clements ML, De Borde D, Maassab HF, Murphy BR. Attenuation of wild-type human influenza A virus by acquisition of the PA polymerase and matrix protein genes of influenza A/Ann Arbor/6/60 cold-adapted donor virus. J Clin Microbiol. 1985. 22:719–725.

43. Carter NJ, Curran MP. Live attenuated influenza vaccine (FluMist[R]; Fluenz): a review of its use in the prevention of seasonal influenza in children and adults. Drugs. 2011. 71:1591–1622.

44. Seo SU, Byun YH, Lee EY, et al. Development and characterization of a live attenuated influenza B virus vaccine candidate. Vaccine. 2008. 26:874–881.

45. Lee KH, Seo SU, Song JM, Lee CM, Kim HA, Seong BL. Characterization of live influenza vaccine donor strain derived from cold-adaptation of X-31 virus. Vaccine. 2006. 24:1966–1974.

46. Desheva JA, Lu XH, Rekstin AR, et al. Characterization of an influenza A H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential. Vaccine. 2006. 24:6859–6866.

47. Treanor JJ, Wilkinson BE, Masseoud F, et al. Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans. Vaccine. 2001. 19:1732–1737.

48. Nicholson KG, Colegate AE, Podda A, et al. Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza. Lancet. 2001. 357:1937–1943.

49. WHO/OIE/FAO H5N1 Evolution Working Group. Continued evolution of highly pathogenic avian influenza A (H5N1): updated nomenclature. Influenza Other Respi Viruses. 2012. 6:1–5.

50. Cohen J. Swine flu pandemic. What's old is new: 1918 virus matches 2009 H1N1 strain. Science. 2010. 327:1563–1564.

51. Xu R, Ekiert DC, Krause JC, Hai R, Crowe JE Jr, Wilson IA. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science. 2010. 328:357–360.

52. Morens DM, Taubenberger JK, Fauci AS. The persistent legacy of the 1918 influenza virus. N Engl J Med. 2009. 361:225–229.

53. Dawood FS, Jain S, et al. Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med. 2009. 360:2605–2615.

54. Centers for Disease Control and Prevention (CDC). 2009 pandemic influenza A (H1N1) virus infections: Chicago, Illinois, April-July 2009. MMWR Morb Mortal Wkly Rep. 2009. 58:913–918.

55. Octaviani CP, Ozawa M, Yamada S, Goto H, Kawaoka Y. High level of genetic compatibility between swine-origin H1N1 and highly pathogenic avian H5N1 influenza viruses. J Virol. 2010. 84:10918–10922.

56. Cline TD, Karlsson EA, Freiden P, et al. Increased pathogenicity of a reassortant 2009 pandemic H1N1 influenza virus containing an H5N1 hemagglutinin. J Virol. 2011. 85:12262–12270.

57. Chen LM, Davis CT, Zhou H, Cox NJ, Donis RO. Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses. PLoS Pathog. 2008. 4:e1000072.

58. Jang YH, Byun YH, Lee YJ, Lee YH, Lee KH, Seong BL. Cold-adapted pandemic 2009 H1N1 influenza virus live vaccine elicits cross-reactive immune responses against seasonal and H5 influenza A viruses. J Virol. 2012. 86:5953–5958.

59. Garten RJ, Davis CT, Russell CA, et al. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science. 2009. 325:197–201.

60. Shinde V, Bridges CB, Uyeki TM, et al. Triple-reassortant swine influenza A (H1) in humans in the United States, 2005-2009. N Engl J Med. 2009. 360:2616–2625.

61. Corti D, Voss J, Gamblin SJ, et al. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science. 2011. 333:850–856.

62. Ekiert DC, Bhabha G, Elsliger MA, et al. Antibody recognition of a highly conserved influenza virus epitope. Science. 2009. 324:246–251.

63. Krause JC, Tsibane T, Tumpey TM, Huffman CJ, Basler CF, Crowe JE Jr. A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin. J Virol. 2011. 85:10905–10908.

64. Prabhu N, Prabakaran M, Ho HT, et al. Monoclonal antibodies against the fusion peptide of hemagglutinin protect mice from lethal influenza A virus H5N1 infection. J Virol. 2009. 83:2553–2562.

65. Sui J, Hwang WC, Perez S, et al. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol. 2009. 16:265–273.

66. Whittle JR, Zhang R, Khurana S, et al. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci U S A. 2011. 108:14216–14221.

67. Ichinohe T, Iwasaki A, Hasegawa H. Innate sensors of influenza virus: clues to developing better intranasal vaccines. Expert Rev Vaccines. 2008. 7:1435–1445.

68. Tamura S, Tanimoto T, Kurata T. Mechanisms of broad cross-protection provided by influenza virus infection and their application to vaccines. Jpn J Infect Dis. 2005. 58:195–207.

69. Shi J, Wen Z, Guo J, et al. Protective efficacy of an H1N1 cold-adapted live vaccine against the 2009 pandemic H1N1, seasonal H1N1, and H5N1 influenza viruses in mice. Antiviral Res. 2012. 93:346–353.

70. Chen Z, Kim L, Subbarao K, Jin H. The 2009 pandemic H1N1 virus induces anti-neuraminidase (NA) antibodies that cross-react with the NA of H5N1 viruses in ferrets. Vaccine. 2012. 30:2516–2522.

71. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A. 2000. 97:6108–6113.

72. Neumann G, Watanabe T, Ito H, et al. Generation of influenza A viruses entirely from cloned cDNAs. Proc Natl Acad Sci U S A. 1999. 96:9345–9350.

73. Jung EJ, Lee KH, Seong BL. Reverse genetic platform for inactivated and live-attenuated influenza vaccine. Exp Mol Med. 2010. 42:116–121.

74. Zhou B, Li Y, Speer SD, Subba A, Lin X, Wentworth DE. Engineering temperature sensitive live attenuated influenza vaccines from emerging viruses. Vaccine. 2012. 30:3691–3702.

75. Geiss GK, Salvatore M, Tumpey TM, et al. Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: the role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza. Proc Natl Acad Sci U S A. 2002. 99:10736–10741.

76. Hayman A, Comely S, Lackenby A, et al. Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway. Virology. 2006. 347:52–64.

77. Min JY, Krug RM. The primary function of RNA binding by the influenza A virus NS1 protein in infected cells: inhibiting the 2'-5' oligo (A) synthetase/RNase L pathway. Proc Natl Acad Sci U S A. 2006. 103:7100–7105.

78. Min JY, Li S, Sen GC, Krug RM. A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis. Virology. 2007. 363:236–243.

79. Egorov A, Brandt S, Sereinig S, et al. Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells. J Virol. 1998. 72:6437–6441.

80. Baskin CR, Bielefeldt-Ohmann H, Garcia-Sastre A, et al. Functional genomic and serological analysis of the protective immune response resulting from vaccination of macaques with an NS1-truncated influenza virus. J Virol. 2007. 81:11817–11827.

81. Hai R, Martinez-Sobrido L, Fraser KA, Ayllon J, García-Sastre A, Palese P. Influenza B virus NS1-truncated mutants: live-attenuated vaccine approach. J Virol. 2008. 82:10580–10590.

82. Falcon AM, Fernandez-Sesma A, Nakaya Y, Moran TM, Ortín J, García-Sastre A. Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins. J Gen Virol. 2005. 86(Pt 10):2817–2821.

83. Wressnigg N, Shurygina AP, Wolff T, et al. Influenza B mutant viruses with truncated NS1 proteins grow efficiently in Vero cells and are immunogenic in mice. J Gen Virol. 2009. 90(Pt 2):366–374.

84. Steel J, Lowen AC, Pena L, et al. Live attenuated influenza viruses containing NS1 truncations as vaccine candidates against H5N1 highly pathogenic avian influenza. J Virol. 2009. 83:1742–1753.

85. Kappes MA, Sandbulte MR, Platt R, et al. Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs. Vaccine. 2012. 30:280–288.

86. Richt JA, Lekcharoensuk P, Lager KM, et al. Vaccination of pigs against swine influenza viruses by using an NS1-truncated modified live-virus vaccine. J Virol. 2006. 80:11009–11018.

87. Quinlivan M, Zamarin D, García-Sastre A, Cullinane A, Chambers T, Palese P. Attenuation of equine influenza viruses through truncations of the NS1 protein. J Virol. 2005. 79:8431–8439.

88. Romanova J, Krenn BM, Wolschek M, et al. Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine. PLoS One. 2009. 4:e5984.

89. Sereinig S, Stukova M, Zabolotnyh N, et al. Influenza virus NS vectors expressing the mycobacterium tuberculosis ESAT-6 protein induce CD4+ Th1 immune response and protect animals against tuberculosis challenge. Clin Vaccine Immunol. 2006. 13:898–904.

90. Stukova MA, Sereinig S, Zabolotnyh NV, et al. Vaccine potential of influenza vectors expressing Mycobacterium tuberculosis ESAT-6 protein. Tuberculosis (Edinb). 2006. 86:236–246.

91. Stech J, Garn H, Wegmann M, Wagner R, Klenk HD. A new approach to an influenza live vaccine: modification of the cleavage site of hemagglutinin. Nat Med. 2005. 11:683–689.

92. Stech J, Garn H, Herwig A, et al. Influenza B virus with modified hemagglutinin cleavage site as a novel attenuated live vaccine. J Infect Dis. 2011. 204:1483–1490.

93. Masic A, Lu X, Li J, et al. Immunogenicity and protective efficacy of an elastase-dependent live attenuated swine influenza virus vaccine administered intranasally in pigs. Vaccine. 2010. 28:7098–7108.

94. Gabriel G, Garn H, Wegmann M, et al. The potential of a protease activation mutant of a highly pathogenic avian influenza virus for a pandemic live vaccine. Vaccine. 2008. 26:956–965.

95. Masic A, Booth JS, Mutwiri GK, Babiuk LA, Zhou Y. Elastase-dependent live attenuated swine influenza A viruses are immunogenic and confer protection against swine influenza A virus infection in pigs. J Virol. 2009. 83:10198–10210.

96. Perez JT, Pham AM, Lorini MH, Chua MA, Steel J, tenOever BR. MicroRNA-mediated species-specific attenuation of influenza A virus. Nat Biotechnol. 2009. 27:572–576.

97. Hui EK, Ralston K, Judd AK, Nayak DP. Conserved cysteine and histidine residues in the putative zinc finger motif of the influenza A virus M1 protein are not critical for influenza virus replication. J Gen Virol. 2003. 84(Pt 11):3105–3113.

98. Hui EK, Smee DF, Wong MH, Nayak DP. Mutations in influenza virus M1 CCHH, the putative zinc finger motif, cause attenuation in mice and protect mice against lethal influenza virus infection. J Virol. 2006. 80:5697–5707.

99. Watanabe S, Watanabe T, Kawaoka Y. Influenza A virus lacking M2 protein as a live attenuated vaccine. J Virol. 2009. 83:5947–5950.

100. Hatta Y, Hatta M, Bilsel P, Neumann G, Kawaoka Y. An M2 cytoplasmic tail mutant as a live attenuated influenza vaccine against pandemic (H1N1) 2009 influenza virus. Vaccine. 2011. 29:2308–2312.

101. Iwatsuki-Horimoto K, Horimoto T, Fujii Y, Kawaoka Y. Generation of influenza A virus NS2 (NEP) mutants with an altered nuclear export signal sequence. J Virol. 2004. 78:10149–10155.

102. Xie H, Liu TM, Lu X, et al. A live attenuated H1N1 M1 mutant provides broad cross-protection against influenza A viruses, including highly pathogenic A/Vietnam/1203/2004, in mice. J Infect Dis. 2009. 200:1874–1883.

103. Coleman JR, Papamichail D, Skiena S, Futcher B, Wimmer E, Mueller S. Virus attenuation by genome-scale changes in codon pair bias. Science. 2008. 320:1784–1787.

104. Mueller S, Coleman JR, Papamichail D, et al. Live attenuated influenza virus vaccines by computer-aided rational design. Nat Biotechnol. 2010. 28:723–726.

105. Manz B, Gotz V, Wunderlich K, et al. Disruption of the viral polymerase complex assembly as a novel approach to attenuate influenza A virus. J Biol Chem. 2011. 286:8414–8424.

106. Li X, Palese P. Mutational analysis of the promoter required for influenza virus virion RNA synthesis. J Virol. 1992. 66:4331–4338.

107. Seong BL, Brownlee GG. A new method for reconstituting influenza polymerase and RNA in vitro: a study of the promoter elements for cRNA and vRNA synthesis in vitro and viral rescue in vivo. Virology. 1992. 186:247–260.

108. Seong BL, Brownlee GG. Nucleotides 9 to 11 of the influenza A virion RNA promoter are crucial for activity in vitro. J Gen Virol. 1992. 73(Pt 12):3115–3124.

109. Fodor E, Pritlove DC, Brownlee GG. The influenza virus panhandle is involved in the initiation of transcription. J Virol. 1994. 68:4092–4096.

110. Catchpole AP, Mingay LJ, Fodor E, Brownlee GG. Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene. J Gen Virol. 2003. 84(Pt 3):507–515.

111. Fodor E, Palese P, Brownlee GG, García-Sastre A. Attenuation of influenza A virus mRNA levels by promoter mutations. J Virol. 1998. 72:6283–6290.

112. Chen Z, Aspelund A, Kemble G, Jin H. Molecular studies of temperature-sensitive replication of the cold-adapted B/Ann Arbor/1/66, the master donor virus for live attenuated influenza FluMist vaccines. Virology. 2008. 380:354–362.

113. Dalton RM, Mullin AE, Amorim MJ, Medcalf E, Tiley LS, Digard P. Temperature sensitive influenza A virus genome replication results from low thermal stability of polymerase-cRNA complexes. Virol J. 2006. 3:58.

114. Poland GA, Ovsyannikova IG, Jacobson RM. Personalized vaccines: the emerging field of vaccinomics. Expert Opin Biol Ther. 2008. 8:1659–1667.

115. Poland GA, Kennedy RB, Ovsyannikova IG. Vaccinomics and personalized vaccinology: is science leading us toward a new path of directed vaccine development and discovery? PLoS Pathog. 2011. 7:e1002344.

116. Minor PD. Vaccines against seasonal and pandemic influenza and the implications of changes in substrates for virus production. Clin Infect Dis. 2010. 50:560–565.

117. Barrett PN, Mundt W, Kistner O, Howard MK. Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines. Expert Rev Vaccines. 2009. 8:607–618.