1887

Abstract

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in children, especially in infants less than 1 year of age. There are currently no licensed vaccines against RSV. rA2ΔM2-2 is a promising live-attenuated vaccine candidate that is currently being evaluated in the clinic. Attenuation of rA2ΔM2-2 is achieved by a single deletion of the M2-2 gene, which disrupts the balance between viral transcription and replication. Whilst performing a manufacturing feasibility study in a serum-free adapted Vero cell line, differences in growth kinetics and cytopathic effect (CPE) were identified between two rA2ΔM2-2 vaccine candidates. Comparative sequence analysis identified four amino acid differences between the two vaccine viruses. Recombinant rA2ΔM2-2 viruses carrying each of the four amino acid differences identified a K66E mutation in the F fragment of the fusion (F) protein as the cause of the growth and CPE differences. Syncytium-formation experiments with RSV F protein carrying mutations at aa 66 suggested that a change in charge at this residue within the F fragment can have a significant impact on fusion.

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2013-12-01
2020-01-24
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References

  1. Bermingham A. , Collins P. L. . ( 1999; ). The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription. . Proc Natl Acad Sci U S A 96:, 11259–11264. [CrossRef] [PubMed]
    [Google Scholar]
  2. Chang A. , Hackett B. , Winter C. C. , Buchholz U. J. , Dutch R. E. . ( 2012; ). Potential electrostatic interactions in multiple regions affect human metapneumovirus F-mediated membrane fusion. . J Virol 86:, 9843–9853. [CrossRef] [PubMed]
    [Google Scholar]
  3. Cheng X. , Zhou H. , Tang R. S. , Munoz M. G. , Jin H. . ( 2001; ). Chimeric subgroup A respiratory syncytial virus with the glycoproteins substituted by those of subgroup B and RSV without the M2-2 gene are attenuated in African green monkeys. . Virology 283:, 59–68. [CrossRef] [PubMed]
    [Google Scholar]
  4. Collins P. L. , Melero J. A. . ( 2011; ). Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. . Virus Res 162:, 80–99. [CrossRef] [PubMed]
    [Google Scholar]
  5. Connors M. , Crowe J. E. Jr , Firestone C.-Y. , Murphy B. R. , Collins P. L. . ( 1995; ). A cold-passaged, attenuated strain of human respiratory syncytial virus contains mutations in the F and L genes. . Virology 208:, 478–484. [CrossRef] [PubMed]
    [Google Scholar]
  6. Crim R. L. , Audet S. A. , Feldman S. A. , Mostowski H. S. , Beeler J. A. . ( 2007; ). Identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity. . J Virol 81:, 261–271. [CrossRef] [PubMed]
    [Google Scholar]
  7. Day N. D. , Branigan P. J. , Liu C. , Gutshall L. L. , Luo J. , Melero J. A. , Sarisky R. T. , Del Vecchio A. M. . ( 2006; ). Contribution of cysteine residues in the extracellular domain of the F protein of human respiratory syncytial virus to its function. . Virol J 3:, 34. [CrossRef] [PubMed]
    [Google Scholar]
  8. Feldman S. A. , Audet S. , Beeler J. A. . ( 2000; ). The fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate. . J Virol 74:, 6442–6447. [CrossRef] [PubMed]
    [Google Scholar]
  9. Gardner A. E. , Dutch R. E. . ( 2007; ). A conserved region in the F(2) subunit of paramyxovirus fusion proteins is involved in fusion regulation. . J Virol 81:, 8303–8314. [CrossRef] [PubMed]
    [Google Scholar]
  10. Hallak L. K. , Spillmann D. , Collins P. L. , Peeples M. E. . ( 2000a; ). Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection. . J Virol 74:, 10508–10513. [CrossRef] [PubMed]
    [Google Scholar]
  11. Hallak L. K. , Collins P. L. , Knudson W. , Peeples M. E. . ( 2000b; ). Iduronic acid-containing glycosaminoglycans on target cells are required for efficient respiratory syncytial virus infection. . Virology 271:, 264–275. [CrossRef] [PubMed]
    [Google Scholar]
  12. Jin H. , Cheng X. , Zhou H. Z. Y. , Li S. , Seddiqui A. . ( 2000; ). Respiratory syncytial virus that lacks open reading frame 2 of the M2 gene (M2-2) has altered growth characteristics and is attenuated in rodents. . J Virol 74:, 74–82. [CrossRef] [PubMed]
    [Google Scholar]
  13. Jin H. , Cheng X. , Traina-Dorge V. L. , Park H. J. , Zhou H. , Soike K. , Kemble G. . ( 2003; ). Evaluation of recombinant respiratory syncytial virus gene deletion mutants in African green monkeys for their potential as live attenuated vaccine candidates. . Vaccine 21:, 3647–3652. [CrossRef] [PubMed]
    [Google Scholar]
  14. Johnson S. , Oliver C. , Prince G. A. , Hemming V. G. , Pfarr D. S. , Wang S. C. , Dormitzer M. , O’Grady J. , Koenig S. . & other authors ( 1997; ). Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. . J Infect Dis 176:, 1215–1224. [CrossRef] [PubMed]
    [Google Scholar]
  15. Kapikian A. Z. , Mitchell R. H. , Chanock R. M. , Shvedoff R. A. , Stewart C. E. . ( 1969; ). An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. . Am J Epidemiol 89:, 405–421.[PubMed]
    [Google Scholar]
  16. Karron R. A. , Wright P. F. , Belshe R. B. , Thumar B. , Casey R. , Newman F. , Polack F. P. , Randolph V. B. , Deatly A. . & other authors ( 2005; ). Identification of a recombinant live attenuated respiratory syncytial virus vaccine candidate that is highly attenuated in infants. . J Infect Dis 191:, 1093–1104. [CrossRef] [PubMed]
    [Google Scholar]
  17. Kaur J. , Tang R. S. , Spaete R. R. , Schickli J. H. . ( 2008; ). Optimization of plasmid-only rescue of highly attenuated and temperature-sensitive respiratory syncytial virus (RSV) vaccine candidates for human trials. . J Virol Methods 153:, 196–202. [CrossRef] [PubMed]
    [Google Scholar]
  18. Kim H. W. , Canchola J. G. , Brandt C. D. , Pyles G. , Chanock R. M. , Jensen K. , Parrott R. H. . ( 1969; ). Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. . Am J Epidemiol 89:, 422–434.[PubMed]
    [Google Scholar]
  19. Martínez I. , Melero J. A. . ( 2000; ). Binding of human respiratory syncytial virus to cells: implication of sulfated cell surface proteoglycans. . J Gen Virol 81:, 2715–2722.[PubMed]
    [Google Scholar]
  20. McLellan J. S. , Yang Y. , Graham B. S. , Kwong P. D. . ( 2011; ). Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. . J Virol 85:, 7788–7796. [CrossRef] [PubMed]
    [Google Scholar]
  21. McLellan J. S. , Chen M. , Leung S. , Graepel K. W. , Du X. , Yang Y. , Zhou T. , Baxa U. , Yasuda E. . & other authors ( 2013; ). Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. . Science 340:, 1113–1117. [CrossRef] [PubMed]
    [Google Scholar]
  22. Nair H. , Nokes D. J. , Gessner B. D. , Dherani M. , Madhi S. A. , Singleton R. J. , O’Brien K. L. , Roca A. , Wright P. F. . & other authors ( 2010; ). Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. . Lancet 375:, 1545–1555. [CrossRef] [PubMed]
    [Google Scholar]
  23. Schickli J. H. , Dubovsky F. , Tang R. S. . ( 2009; ). Challenges in developing a pediatric RSV vaccine. . Hum Vaccin 5:, 582–591.[PubMed] [CrossRef]
    [Google Scholar]
  24. Schlender J. , Zimmer G. , Herrler G. , Conzelmann K. K. . ( 2003; ). Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G, determines the specificity of RSV infection. . J Virol 77:, 4609–4616. [CrossRef] [PubMed]
    [Google Scholar]
  25. Tayyari F. , Marchant D. , Moraes T. J. , Duan W. , Mastrangelo P. , Hegele R. G. . ( 2011; ). Identification of nucleolin as a cellular receptor for human respiratory syncytial virus. . Nat Med 17:, 1132–1135. [CrossRef] [PubMed]
    [Google Scholar]
  26. Teng M. N. , Collins P. L. . ( 1999; ). Altered growth characteristics of recombinant respiratory syncytial viruses which do not produce NS2 protein. . J Virol 73:, 466–473.[PubMed]
    [Google Scholar]
  27. Teng M. N. , Whitehead S. S. , Bermingham A. , St Claire M. , Elkins W. R. , Murphy B. R. , Collins P. L. . ( 2000; ). Recombinant respiratory syncytial virus that does not express the NS1 or M2-2 protein is highly attenuated and immunogenic in chimpanzees. . J Virol 74:, 9317–9321. [CrossRef] [PubMed]
    [Google Scholar]
  28. Teng M. N. , Whitehead S. S. , Collins P. L. . ( 2001; ). Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo. . Virology 289:, 283–296. [CrossRef] [PubMed]
    [Google Scholar]
  29. Whitehead S. S. , Juhasz K. , Firestone C.-Y. , Collins P. L. , Murphy B. R. . ( 1998; ). Recombinant respiratory syncytial virus (RSV) bearing a set of mutations from cold-passaged RSV is attenuated in chimpanzees. . J Virol 72:, 4467–4471.[PubMed]
    [Google Scholar]
  30. Wright P. F. , Karron R. A. , Belshe R. B. , Shi J. R. , Randolph V. B. , Collins P. L. , O’Shea A. F. , Gruber W. C. , Murphy B. R. . ( 2007; ). The absence of enhanced disease with wild type respiratory syncytial virus infection occurring after receipt of live, attenuated, respiratory syncytial virus vaccines. . Vaccine 25:, 7372–7378. [CrossRef] [PubMed]
    [Google Scholar]
  31. Wu H. , Pfarr D. S. , Johnson S. , Brewah Y. A. , Woods R. M. , Patel N. K. , White W. I. , Young J. F. , Kiener P. A. . ( 2007; ). Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. . J Mol Biol 368:, 652–665. [CrossRef] [PubMed]
    [Google Scholar]
  32. Yuk I. H. , Lin G. B. , Ju H. , Sifi I. , Lam Y. , Cortez A. , Liebertz D. , Berry J. M. , Schwartz R. M. . ( 2006; ). A serum-free Vero production platform for a chimeric virus vaccine candidate. . Cytotechnology 51:, 183–192. [CrossRef] [PubMed]
    [Google Scholar]
  33. Zhu Q. , Patel N. K. , McAuliffe J. M. , Zhu W. , Wachter L. , McCarthy M. P. , Suzich J. A. . ( 2012; ). Natural polymorphisms and resistance-associated mutations in the fusion protein of respiratory syncytial virus (RSV): effects on RSV susceptibility to palivizumab. . J Infect Dis 205:, 635–638. [CrossRef] [PubMed]
    [Google Scholar]
  34. Zimmer G. , Trotz I. , Herrler G. . ( 2001; ). N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus. . J Virol 75:, 4744–4751. [CrossRef] [PubMed]
    [Google Scholar]
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