Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement

Abstract

Adenovirus type 5 (Ad5) specifically binds coagulation factor X (FX), and FX is normally essential for intravenously injected Ad5 vectors to transduce the liver. We demonstrate that the ability of FX to enhance liver transduction by Ad5 vectors is due to an unexpected ability of FX to protect Ad5 from attack by the classical complement pathway. In vitro, naive mouse serum neutralized Ad5 when FX was blocked from binding Ad5. This neutralization was mediated by natural IgM and the classical complement pathway. In vivo, FX was essential for Ad5 vectors to transduce the livers of wild-type mice, but FX was not required for liver transduction in mice that lack antibodies, C1q or C4. We conclude that Ad5 recruits FX as a defense against complement and that the sensitivity of Ad5 to inactivation by complement must be taken into account when designing vectors for systemic gene therapy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Vitamin K–dependent coagulation factors are required for liver transduction in WT C57BL/6 mice but not in Rag1−/− (B and T cell–deficient) mice.
Figure 2: B cells and natural IgM determine whether coagulation factors are required for liver transduction.
Figure 3: The classical complement pathway restricts liver transduction when coagulation factors are absent.
Figure 4: FX in mouse serum shields Ad5 from neutralization by complement.
Figure 5: FX blocks the ability of Ad5 vectors to activate C3.
Figure 6: Natural antibodies and complement inhibit transduction of nonhepatic organs when Ad5 vectors are unprotected by coagulation factors.

Similar content being viewed by others

References

  1. Cichon, G. et al. Complement activation by recombinant adenoviruses. Gene Ther. 8, 1794–1800 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Jiang, H., Wang, Z., Serra, D., Frank, M.M. & Amalfitano, A. Recombinant adenovirus vectors activate the alternative complement pathway, leading to the binding of human complement protein C3 independent of anti-Ad antibodies. Mol. Ther. 10, 1140–1142 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Shayakhmetov, D.M., Gaggar, A., Ni, S., Li, Z.Y. & Lieber, A. Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity. J. Virol. 79, 7478–7491 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Perreau, M., Guerin, M.C., Drouet, C. & Kremer, E.J. Interactions between human plasma components and a xenogenic adenovirus vector: reduced immunogenicity during gene transfer. Mol. Ther. 15, 1998–2007 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Xu, Z., Tian, J., Smith, J.S. & Byrnes, A.P. Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies and complement. J. Virol. 82, 11705–11713 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Carlisle, R.C. et al. Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1. Blood 113, 1909–1918 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Tian, J. et al. Adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo. J. Virol. 83, 5648–5658 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ochsenbein, A.F. et al. Control of early viral and bacterial distribution and disease by natural antibodies. Science 286, 2156–2159 (1999).

    Article  CAS  PubMed  Google Scholar 

  9. Notkins, A.L. Polyreactivity of antibody molecules. Trends Immunol. 25, 174–179 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Racine, R. & Winslow, G.M. IgM in microbial infections: taken for granted? Immunol. Lett. 125, 79–85 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rambach, G., Wurzner, R. & Speth, C. Complement: an efficient sword of innate immunity. Contrib. Microbiol. 15, 78–100 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. He, J.Q. et al. CRIg mediates early Kupffer cell responses to adenovirus. J. Leukoc. Biol. 93, 301–306 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Kalyuzhniy, O. et al. Adenovirus serotype 5 hexon is critical for virus infection of hepatocytes in vivo. Proc. Natl. Acad. Sci. USA 105, 5483–5488 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Waddington, S.N. et al. Adenovirus serotype 5 hexon mediates liver gene transfer. Cell 132, 397–409 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Doronin, K. et al. Coagulation factor X activates innate immunity to human species C adenovirus. Science 338, 795–798 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Parker, A.L. et al. Multiple Vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes in vitro and in vivo. Blood 108, 2554–2561 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Vigant, F. et al. Substitution of hexon hypervariable region 5 of adenovirus serotype 5 abrogates blood factor binding and limits gene transfer to liver. Mol. Ther. 16, 1474–1480 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Alba, R. et al. Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: effect of mutagenesis on FX interactions and gene transfer. Blood 114, 965–971 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jonsson, M.I. et al. Coagulation factors IX and X enhance binding and infection of adenovirus types 5 and 31 in human epithelial cells. J. Virol. 83, 3816–3825 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bradshaw, A.C. et al. Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X. PLoS Pathog. 6, e1001142 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Duffy, M.R., Bradshaw, A.C., Parker, A.L., McVey, J.H. & Baker, A.H. A cluster of basic amino acids in the factor X serine protease mediate surface attachment of adenovirus:FX complexes. J. Virol. 85, 10914–10919 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Coughlan, L. et al. Tropism-modification strategies for targeted gene delivery using adenoviral vectors. Viruses 2, 2290–2355 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Mombaerts, P. et al. Rag1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 (1992).

    Article  CAS  PubMed  Google Scholar 

  24. Atoda, H., Ishikawa, M., Mizuno, H. & Morita, T. Coagulation factor X-binding protein from Deinagkistrodon acutus venom is a Gla domain-binding protein. Biochemistry 37, 17361–17370 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Campos, S.K. & Barry, M.A. Rapid construction of capsid-modified adenoviral vectors through bacteriophage lambda Red recombination. Hum. Gene Ther. 15, 1125–1130 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Smith, T.A. et al. Adenovirus serotype 5 fiber shaft influences in vivo gene transfer in mice. Hum. Gene Ther. 14, 777–787 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Klein-Schneegans, A.S., Kuntz, L., Trembleau, S., Fonteneau, P. & Loor, F. Serum concentrations of IgM, IgG1, IgG2b, IgG3 and IgA in C57BL/6 mice and their congenics at the nu (nude) locus. Thymus 16, 45–54 (1990).

    CAS  PubMed  Google Scholar 

  28. Chen, J. et al. Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus. Int. Immunol. 5, 647–656 (1993).

    Article  CAS  PubMed  Google Scholar 

  29. Hannum, L.G., Haberman, A.M., Anderson, S.M. & Shlomchik, M.J. Germinal center initiation, variable gene region hypermutation, and mutant B cell selection without detectable immune complexes on follicular dendritic cells. J. Exp. Med. 192, 931–942 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Khare, R., Reddy, V.S., Nemerow, G.R. & Barry, M.A. Identification of adenovirus serotype 5 hexon regions that interact with scavenger receptors. J. Virol. 86, 2293–2301 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Alba, R. et al. Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors. Blood 116, 2656–2664 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Amara, U. et al. Molecular intercommunication between the complement and coagulation systems. J. Immunol. 185, 5628–5636 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Beebe, D.P. & Cooper, N.R. Neutralization of vesicular stomatitis virus (VSV) by human complement requires a natural IgM antibody present in human serum. J. Immunol. 126, 1562–1568 (1981).

    CAS  PubMed  Google Scholar 

  34. Rother, R.P. et al. A novel mechanism of retrovirus inactivation in human serum mediated by anti-alpha-galactosyl natural antibody. J. Exp. Med. 182, 1345–1355 (1995).

    Article  CAS  PubMed  Google Scholar 

  35. DePolo, N.J. et al. VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum. Mol. Ther. 2, 218–222 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Sokoloff, A.V. et al. Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum. Mol. Ther. 3, 821–830 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Wakimoto, H. et al. The complement response against an oncolytic virus is species-specific in its activation pathways. Mol. Ther. 5, 275–282 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Hoare, J., Waddington, S., Thomas, H.C., Coutelle, C. & McGarvey, M.J. Complement inhibition rescued mice allowing observation of transgene expression following intraportal delivery of baculovirus in mice. J. Gene Med. 7, 325–333 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Jayasekera, J.P., Moseman, E.A. & Carroll, M.C. Natural antibody and complement mediate neutralization of influenza virus in the absence of prior immunity. J. Virol. 81, 3487–3494 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Moulton, E.A., Atkinson, J.P. & Buller, R.M. Surviving mousepox infection requires the complement system. PLoS Pathog. 4, e1000249 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Lambris, J.D., Ricklin, D. & Geisbrecht, B.V. Complement evasion by human pathogens. Nat. Rev. Microbiol. 6, 132–142 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Stoermer, K.A. & Morrison, T.E. Complement and viral pathogenesis. Virology 411, 362–373 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Carlsson, F., Sandin, C. & Lindahl, G. Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway. Mol. Microbiol. 56, 28–39 (2005).

    Article  CAS  PubMed  Google Scholar 

  44. Prill, J.M. et al. Modifications of adenovirus hexon allow for either hepatocyte detargeting or targeting with potential evasion from Kupffer cells. Mol. Ther. 19, 83–92 (2011).

    Article  CAS  PubMed  Google Scholar 

  45. Shayakhmetov, D.M., Li, Z.Y., Ni, S. & Lieber, A. Analysis of adenovirus sequestration in the liver, transduction of hepatic cells, and innate toxicity after injection of fiber-modified vectors. J. Virol. 78, 5368–5381 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Xu, Z.L. et al. Strength evaluation of transcriptional regulatory elements for transgene expression by adenovirus vector. J. Control. Release 81, 155–163 (2002).

    Article  CAS  PubMed  Google Scholar 

  47. Smith, J.S., Tian, J., Muller, J. & Byrnes, A.P. Unexpected pulmonary uptake of adenovirus vectors in animals with chronic liver disease. Gene Ther. 11, 431–438 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Manickan, E. et al. Rapid Kupffer cell death after intravenous injection of adenovirus vectors. Mol. Ther. 13, 108–117 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Förster, I. & Rajewsky, K. Expansion and functional activity of Ly-1+ B cells upon transfer of peritoneal cells into allotype-congenic, newborn mice. Eur. J. Immunol. 17, 521–528 (1987).

    Article  PubMed  Google Scholar 

  50. Lachmann, P.J. Preparing serum for functional complement assays. J. Immunol. Methods 352, 195–197 (2010).

    Article  CAS  PubMed  Google Scholar 

  51. Kettner, C. & Shaw, E. D-Phe-Pro-ArgCH2C1-A selective affinity label for thrombin. Thromb. Res. 14, 969–973 (1979).

    Article  CAS  PubMed  Google Scholar 

  52. McCarthy, D.A. & Macey, M.G. Novel anticoagulants for flow cytometric analysis of live leucocytes in whole blood. Cytometry 23, 196–204 (1996).

    Article  CAS  PubMed  Google Scholar 

  53. Kettner, C. & Shaw, E. The selective affinity labeling of factor Xa by peptides of arginine chloromethyl ketone. Thromb. Res. 22, 645–652 (1981).

    Article  CAS  PubMed  Google Scholar 

  54. Wiethoff, C.M., Wodrich, H., Gerace, L. & Nemerow, G.R. Adenovirus protein VI mediates membrane disruption following capsid disassembly. J. Virol. 79, 1992–2000 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Funding was provided by the US Food and Drug Administration (FDA), including the FDA's Critical Path program. This project was supported in part by fellowships administered by the Oak Ridge Institute for Science and Education. We thank M. Barry (Mayo Clinic) for providing vectors, H. Mizuguchi (Osaka University) for providing plasmid pAdHM4-CMVL1 and M. Diamond (Washington University) for providing C1qa−/− mice. We thank M. Shlomchik (Yale University), S. Epstein (FDA) and J. Misplon (FDA) for providing JHD and mIg Tg mice. We thank the Center for Biologics Evaluation and Research animal facility staff for outstanding support. We thank S. Epstein, C. Kimchi-Sarfaty, G. Price and C. Wiethoff for helpful discussions or comments on the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

Z.X. discovered that coagulation factors are not required for liver transduction. A.P.B., Z.X., Q.Q., J.T. and J.S.S. designed experiments. Z.X. performed all in vivo experiments, collected serum and plasma, purified IgM, constructed AdHVR7, grew vectors, and performed quantitative PCR, western blotting and Octet RED assays. Q.Q. performed quantitative PCR and conducted in vitro transduction and neutralization studies. J.T. conducted complement activity assays and grew vectors. J.S.S. conducted cell-binding assays. G.M.C. conducted transduction assays. T.M. purified X-bp. A.P.B. bred mice. A.P.B. wrote the majority of the manuscript, and all authors participated in the preparation of the manuscript.

Corresponding author

Correspondence to Andrew P Byrnes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Tables 1 and 2 (PDF 688 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xu, Z., Qiu, Q., Tian, J. et al. Coagulation factor X shields adenovirus type 5 from attack by natural antibodies and complement. Nat Med 19, 452–457 (2013). https://doi.org/10.1038/nm.3107

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3107

This article is cited by

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research