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.

  • Original Article
  • Published:

Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines

Gene Therapy volume 16, pages 359–366 (2009)Cite this article

Abstract

Replicon plasmids encoding an alphavirus RNA replicase constitute an alternative to conventional DNA plasmids with promise for DNA vaccination in humans. Replicase activity amplifies the levels of transgene mRNA through a copying process involving double-stranded (ds) RNA intermediates, which contribute to vaccine immunogenicity by activating innate antiviral responses. Toll-like receptor 3 (TLR3) is a dsRNA innate immune receptor expressed by antigen-presenting dendritic cells (DCs). Here, we test the hypothesis that TLR3 is necessary for the immunogenicity of replicon plasmid-based DNA vaccines. We show that mouse CD8α+ DC phagocytose dying replicon plasmid-transfected cells in vitro and are activated in a TLR3-dependent manner by dsRNA present within those cells. However, we find that cytotoxic T-cell responses to a replicon plasmid intramuscular vaccine are not diminished in the absence of TLR3 in vivo. Our results underscore the potential role of TLR3 in mediating immune activation by dsRNA-bearing replicon plasmid-transfected cells and indicate that other innate sensing pathways can compensate for TLR3 absence in vivo.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Greenland JR, Letvin NL . Chemical adjuvants for plasmid DNA vaccines. Vaccine 2007; 25: 3731–3741.

    Article  CAS  PubMed  Google Scholar 

  2. Forde GM . Rapid-response vaccines—does DNA offer a solution? Nat Biotechnol 2005; 23: 1059–1062.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lu S, Wang S, Grimes-Serrano JM . Current progress of DNA vaccine studies in humans. Expert Rev Vaccines 2008; 7: 175–191.

    Article  CAS  PubMed  Google Scholar 

  4. Dupuis M, Denis-Mize K, Woo C, Goldbeck C, Selby MJ, Chen M et al. Distribution of DNA vaccines determines their immunogenicity after intramuscular injection in mice. J Immunol 2000; 165: 2850–2858.

    Article  CAS  PubMed  Google Scholar 

  5. Lundstrom K . Alphavirus-based vaccines. Curr Opin Mol Ther 2002; 4: 28–34.

    CAS  PubMed  Google Scholar 

  6. Driver DA, Latham EM, Polo JM, Belli BA, Banks TA, Chada S et al. Layered amplification of gene expression with a DNA gene delivery system. Ann N Y Acad Sci 1995; 772: 261–264.

    Article  CAS  PubMed  Google Scholar 

  7. Herweijer H, Latendresse JS, Williams P, Zhang G, Danko I, Schlesinger S et al. A plasmid-based self-amplifying Sindbis virus vector. Hum Gene Ther 1995; 6: 1161–1167.

    Article  CAS  PubMed  Google Scholar 

  8. Leitner WW, Hwang LN, deVeer MJ, Zhou A, Silverman RH, Williams BR et al. Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways. Nat Med 2003; 9: 33–39.

    Article  CAS  PubMed  Google Scholar 

  9. Leitner WW, Bergmann-Leitner ES, Hwang LN, Restifo NP . Type I interferons are essential for the efficacy of replicase-based DNA vaccines. Vaccine 2006; 24: 5110–5118.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Smerdou C, Liljestrom P . Non-viral amplification systems for gene transfer: vectors based on alphaviruses. Curr Opin Mol Ther 1999; 1: 244–251.

    CAS  PubMed  Google Scholar 

  11. Pichlmair A, Reis e Sousa C . Innate recognition of viruses. Immunity 2007; 27: 370–383.

    Article  CAS  PubMed  Google Scholar 

  12. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA . Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 2001; 413: 732–738.

    Article  CAS  PubMed  Google Scholar 

  13. Matsumoto M, Kikkawa S, Kohase M, Miyake K, Seya T . Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem Biophys Res Commun 2002; 293: 1364–1369.

    Article  CAS  PubMed  Google Scholar 

  14. Oshiumi H, Matsumoto M, Funami K, Akazawa T, Seya T . TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat Immunol 2003; 4: 161–167.

    Article  CAS  PubMed  Google Scholar 

  15. Edwards AD, Diebold SS, Slack EM, Tomizawa H, Hemmi H, Kaisho T et al. Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8 alpha+ DC correlates with unresponsiveness to imidazoquinolines. Eur J Immunol 2003; 33: 827–833.

    Article  CAS  PubMed  Google Scholar 

  16. Schulz O, Diebold SS, Chen M, Naslund TI, Nolte MA, Alexopoulou L et al. Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature 2005; 433: 887–892.

    Article  CAS  PubMed  Google Scholar 

  17. Leitner WW, Ying H, Driver DA, Dubensky TW, Restifo NP . Enhancement of tumor-specific immune response with plasmid DNA replicon vectors. Cancer Res 2000; 60: 51–55.

    CAS  PubMed Central  PubMed  Google Scholar 

  18. Leitner WW, Hwang LN, Bergmann-Leitner ES, Finkelstein SE, Frank S, Restifo NP . Apoptosis is essential for the increased efficacy of alphaviral replicase-based DNA vaccines. Vaccine 2004; 22: 1537–1544.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Schulz O, Reis e Sousa C . Cross-presentation of cell-associated antigens by CD8alpha+ dendritic cells is attributable to their ability to internalize dead cells. Immunology 2002; 107: 183–189.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Villadangos JA, Schnorrer P . Intrinsic and cooperative antigen-presenting functions of dendritic-cell subsets in vivo. Nat Rev Immunol 2007; 7: 543–555.

    Article  CAS  PubMed  Google Scholar 

  21. Le Bon A, Et N, Rossmann C, Ashton M, Hou S, Gewert D et al. Cross-priming of CD8+ T cells stimulated by virus-induced type I interferon. Nat Immunol 2003; 4: 1009–1015.

    Article  CAS  PubMed  Google Scholar 

  22. Condon C, Watkins SC, Celluzzi CM, Thompson K, Falo Jr LD . DNA-based immunization by in vivo transfection of dendritic cells. Nat Med 1996; 2: 1122–1128.

    Article  CAS  PubMed  Google Scholar 

  23. Casares S, Inaba K, Brumeanu TD, Steinman RM, Bona CA . Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope. J Exp Med 1997; 186: 1481–1486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Porgador A, Irvine KR, Iwasaki A, Barber BH, Restifo NP, Germain RN . Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization. J Exp Med 1998; 188: 1075–1082.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Spies B, Hochrein H, Vabulas M, Huster K, Busch DH, Schmitz F et al. Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice. J Immunol 2003; 171: 5908–5912.

    Article  CAS  PubMed  Google Scholar 

  26. Babiuk S, Mookherjee N, Pontarollo R, Griebel P, van Drunen Littel-van den Hurk S, Hecker R et al. TLR9−/− and TLR9+/+ mice display similar immune responses to a DNA vaccine. Immunology 2004; 113: 114–120.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Malathi K, Dong B, Gale Jr M, Silverman RH . Small self-RNA generated by RNase L amplifies antiviral innate immunity. Nature 2007; 448: 816–819.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 2004; 5: 730–737.

    Article  CAS  PubMed  Google Scholar 

  29. Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 2006; 441: 101–105.

    Article  CAS  PubMed  Google Scholar 

  30. Gitlin L, Barchet W, Gilfillan S, Cella M, Beutler B, Flavell RA et al. Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus. Proc Natl Acad Sci USA 2006; 103: 8459–8464.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 2007; 448: 501–505.

    Article  CAS  PubMed  Google Scholar 

  32. Kato H, Takeuchi O, Mikamo-Satoh E, Hirai R, Kawai T, Matsushita K et al. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5. J Exp Med 2008; 205: 1601–1610.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ishii KJ, Kawagoe T, Koyama S, Matsui K, Kumar H, Kawai T et al. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines. Nature 2008; 451: 725–729.

    Article  CAS  PubMed  Google Scholar 

  34. Schulz O, Edwards AD, Schito M, Aliberti J, Manickasingham S, Sher A et al. CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity 2000; 13: 453–462.

    Article  CAS  PubMed  Google Scholar 

  35. Diebold SS, Cotten M, Koch N, Zenke M . MHC class II presentation of endogenously expressed antigens by transfected dendritic cells. Gene Therapy 2001; 8: 487–493.

    Article  CAS  PubMed  Google Scholar 

  36. van den Hoff MJ, Moorman AF, Lamers WH . Electroporation in ‘intracellular’ buffer increases cell survival. Nucleic Acids Res 1992; 20: 2902.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Vincenzo Cerundolo and Michael Palmowski for their kind advice on DNA vaccination and members of the Immunobiology Laboratory, CRUK, for support and useful discussions. SSD is currently funded by a CRUK Career Development Award.

Author information

Author notes

  1. S S Diebold

    Present address: 5Current address: King's College London, Peter Gorer Department of Immunobiology, Guy's Hospital, London SE1 9RT, UK,

  2. S S Diebold and O Schulz: These authors contributed equally to this work.

Authors and Affiliations

  1. Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK

    S S Diebold, O Schulz & C Reis e Sousa

  2. Centre d'Immunologie de Marseille-Luminy, CNRS-INSERM-Université de la Méditerranée, Parc Scientifique de Luminy, Marseille, France

    L Alexopoulou

  3. National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    W W Leitner

  4. Section of Immunobiology, Yale University School of Medicine and Howard Hughes Medical Insitute, New Haven, CT, USA

    R A Flavell

Authors
  1. S S Diebold
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. O Schulz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L Alexopoulou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. W W Leitner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. R A Flavell
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C Reis e Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S S Diebold.

Additional information

Conflict of interest

The authors state no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Diebold, S., Schulz, O., Alexopoulou, L. et al. Role of TLR3 in the immunogenicity of replicon plasmid-based vaccines. Gene Ther 16, 359–366 (2009). https://doi.org/10.1038/gt.2008.164

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2008.164

Keywords

This article is cited by

Search

Advanced search

Quick links