1887

Abstract

The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.

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2014-02-01
2024-12-06
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References

  1. Alonso-Caplen F. V., Nemeroff M. E., Qiu Y., Krug R. M. 1992; Nucleocytoplasmic transport: the influenza virus NS1 protein regulates the transport of spliced NS2 mRNA and its precursor NS1 mRNA. Genes Dev 6:255–267 [View Article][PubMed]
    [Google Scholar]
  2. Bullido R., Gómez-Puertas P., Saiz M. J., Portela A. 2001; Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs. J Virol 75:4912–4917 [View Article][PubMed]
    [Google Scholar]
  3. Egorov A., Brandt S., Sereinig S., Romanova J., Ferko B., Katinger D., Grassauer A., Alexandrova G., Katinger H., Muster T. 1998; Transfectant influenza A viruses with long deletions in the NS1 protein grow efficiently in Vero cells. J Virol 72:6437–6441[PubMed]
    [Google Scholar]
  4. Ferko B., Stasakova J., Sereinig S., Romanova J., Katinger D., Niebler B., Katinger H., Egorov A. 2001; Hyperattenuated recombinant influenza A virus nonstructural-protein-encoding vectors induce human immunodeficiency virus type 1 Nef-specific systemic and mucosal immune responses in mice. J Virol 75:8899–8908 [View Article][PubMed]
    [Google Scholar]
  5. García-Sastre A. 2000; Transfectant influenza viruses as antigen delivery vectors. Adv Virus Res 55:579–597 [View Article][PubMed]
    [Google Scholar]
  6. García-Sastre A., Palese P. 1995; Influenza virus vectors. Biologicals 23:171–178 [View Article][PubMed]
    [Google Scholar]
  7. García-Sastre A., Egorov A., Matassov D., Brandt S., Levy D. E., Durbin J. E., Palese P., Muster T. 1998; Influenza A virus lacking the NS1 gene replicates in interferon-deficient systems. Virology 252:324–330 [View Article][PubMed]
    [Google Scholar]
  8. He Q., Martinez-Sobrido L., Eko F. O., Palese P., Garcia-Sastre A., Lyn D., Okenu D., Bandea C., Ananaba G. A.other authors 2007; Live-attenuated influenza viruses as delivery vectors for Chlamydia vaccines. Immunology 122:28–37 [View Article][PubMed]
    [Google Scholar]
  9. Heaton N. S., Leyva-Grado V. H., Tan G. S., Eggink D., Hai R., Palese P. 2013; In vivo bioluminescent imaging of influenza a virus infection and characterization of novel cross-protective monoclonal antibodies. J Virol 87:8272–8281 [View Article][PubMed]
    [Google Scholar]
  10. Hoffmann E., Webster R. G. 2000; Unidirectional RNA polymerase I-polymerase II transcription system for the generation of influenza A virus from eight plasmids. J Gen Virol 81:2843–2847[PubMed]
    [Google Scholar]
  11. Hoffmann E., Neumann G., Kawaoka Y., Hobom G., Webster R. G. 2000; A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97:6108–6113 [View Article][PubMed]
    [Google Scholar]
  12. Hoffmann E., Stech J., Guan Y., Webster R. G., Perez D. R. 2001; Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146:2275–2289 [View Article][PubMed]
    [Google Scholar]
  13. Kittel C., Sereinig S., Ferko B., Stasakova J., Romanova J., Wolkerstorfer A., Katinger H., Egorov A. 2004; Rescue of influenza virus expressing GFP from the NS1 reading frame. Virology 324:67–73 [View Article][PubMed]
    [Google Scholar]
  14. Kittel C., Ferko B., Kurz M., Voglauer R., Sereinig S., Romanova J., Stiegler G., Katinger H., Egorov A. 2005; Generation of an influenza A virus vector expressing biologically active human interleukin-2 from the NS gene segment. J Virol 79:10672–10677 [View Article][PubMed]
    [Google Scholar]
  15. Langley W. A., Bradley K. C., Li Z. N., Smith M. E., Schnell M. J., Steinhauer D. A. 2010; Induction of neutralizing antibody responses to anthrax protective antigen by using influenza virus vectors: implications for disparate immune system priming pathways. J Virol 84:8300–8307 [View Article][PubMed]
    [Google Scholar]
  16. Manicassamy B., Manicassamy S., Belicha-Villanueva A., Pisanelli G., Pulendran B., García-Sastre A. 2010; Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus. Proc Natl Acad Sci U S A 107:11531–11536 [View Article][PubMed]
    [Google Scholar]
  17. Mänz B., Brunotte L., Reuther P., Schwemmle M. 2012; Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells. Nat Commun 3:802 [View Article][PubMed]
    [Google Scholar]
  18. Muster T., Rajtarova J., Sachet M., Unger H., Fleischhacker R., Romirer I., Grassauer A., Url A., García-Sastre A.other authors 2004; Interferon resistance promotes oncolysis by influenza virus NS1-deletion mutants. Int J Cancer 110:15–21 [View Article][PubMed]
    [Google Scholar]
  19. Neumann G., Kawaoka Y. 2002; Generation of influenza A virus from cloned cDNAs–historical perspective and outlook for the new millenium. Rev Med Virol 12:13–30 [View Article][PubMed]
    [Google Scholar]
  20. Neumann G., Whitt M. A., Kawaoka Y. 2002; A decade after the generation of a negative-sense RNA virus from cloned cDNA – what have we learned?. J Gen Virol 83:2635–2662[PubMed]
    [Google Scholar]
  21. O’Neill R. E., Talon J., Palese P. 1998; The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins. EMBO J 17:288–296 [View Article][PubMed]
    [Google Scholar]
  22. Ozawa M., Victor S. T., Taft A. S., Yamada S., Li C., Hatta M., Das S. C., Takashita E., Kakugawa S.other authors 2011; Replication-incompetent influenza A viruses that stably express a foreign gene. J Gen Virol 92:2879–2888 [View Article][PubMed]
    [Google Scholar]
  23. Palese P., Muster T., Zheng H., O’Neill R., Garcia-Sastre A. 1999; Learning from our foes: a novel vaccine concept for influenza virus. Arch Virol Suppl 15:131–138[PubMed]
    [Google Scholar]
  24. Paterson D., Fodor E. 2012; Emerging roles for the influenza A virus nuclear export protein (NEP). PLoS Pathog 8:e1003019 [View Article][PubMed]
    [Google Scholar]
  25. Pena L., Vincent A. L., Ye J., Ciacci-Zanella J. R., Angel M., Lorusso A., Gauger P. C., Janke B. H., Loving C. L., Perez D. R. 2011; Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses. J Virol 85:456–469 [View Article][PubMed]
    [Google Scholar]
  26. Pena L., Sutton T., Chockalingam A., Kumar S., Angel M., Shao H., Chen H., Li W., Perez D. R. 2013; Influenza viruses with rearranged genomes as live-attenuated vaccines. J Virol 87:5118–5127 [View Article][PubMed]
    [Google Scholar]
  27. Percy N., Barclay W. S., García-Sastre A., Palese P. 1994; Expression of a foreign protein by influenza A virus. J Virol 68:4486–4492[PubMed]
    [Google Scholar]
  28. Read E. K., Digard P. 2010; Individual influenza A virus mRNAs show differential dependence on cellular NXF1/TAP for their nuclear export. J Gen Virol 91:1290–1301 [View Article][PubMed]
    [Google Scholar]
  29. Rimmelzwaan G. F., Verburgh R. J., Nieuwkoop N. J., Bestebroer T. M., Fouchier R. A., Osterhaus A. D. 2011; Use of GFP-expressing influenza viruses for the detection of influenza virus A/H5N1 neutralizing antibodies. Vaccine 29:3424–3430 [View Article][PubMed]
    [Google Scholar]
  30. Robb N. C., Jackson D., Vreede F. T., Fodor E. 2010; Splicing of influenza A virus NS1 mRNA is independent of the viral NS1 protein. J Gen Virol 91:2331–2340 [View Article][PubMed]
    [Google Scholar]
  31. Sereinig S., Stukova M., Zabolotnyh N., Ferko B., Kittel C., Romanova J., Vinogradova T., Katinger H., Kiselev O., Egorov A. 2006; 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 13:898–904 [View Article][PubMed]
    [Google Scholar]
  32. Skeiky Y. A., Sadoff J. C. 2006; Advances in tuberculosis vaccine strategies. Nat Rev Microbiol 4:469–476 [View Article][PubMed]
    [Google Scholar]
  33. Song H., Nieto G. R., Perez D. R. 2007; A new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates. J Virol 81:9238–9248 [View Article][PubMed]
    [Google Scholar]
  34. Sutejo R., Yeo D. S., Myaing M. Z., Hui C., Xia J., Ko D., Cheung P. C., Tan B. H., Sugrue R. J. 2012; Activation of type I and III interferon signalling pathways occurs in lung epithelial cells infected with low pathogenic avian influenza viruses. PLoS ONE 7:e33732 [View Article][PubMed]
    [Google Scholar]
  35. Talon J., Salvatore M., O’Neill R. E., Nakaya Y., Zheng H., Muster T., García-Sastre A., Palese P. 2000; Influenza A and B viruses expressing altered NS1 proteins: a vaccine approach. Proc Natl Acad Sci U S A 97:4309–4314 [View Article][PubMed]
    [Google Scholar]
  36. Wacheck V., Egorov A., Groiss F., Pfeiffer A., Fuereder T., Hoeflmayer D., Kundi M., Popow-Kraupp T., Redlberger-Fritz M.other authors 2010; A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist NS1. J Infect Dis 201:354–362 [View Article][PubMed]
    [Google Scholar]
  37. Wang L., Suarez D. L., Pantin-Jackwood M., Mibayashi M., García-Sastre A., Saif Y. M., Lee C. W. 2008; Characterization of influenza virus variants with different sizes of the non-structural (NS) genes and their potential as a live influenza vaccine in poultry. Vaccine 26:3580–3586 [View Article][PubMed]
    [Google Scholar]
  38. Wolschek M., Samm E., Seper H., Sturlan S., Kuznetsova I., Schwager C., Khassidov A., Kittel C., Muster T.other authors 2011; Establishment of a chimeric, replication-deficient influenza A virus vector by modulation of splicing efficiency. J Virol 85:2469–2473 [View Article][PubMed]
    [Google Scholar]
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