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Journal of Virology, January 2007, p. 539-547, Vol. 81, No. 2
0022-538X/07/$08.00+0     doi:10.1128/JVI.01818-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Tubulovesicular Structures within Vesicular Stomatitis Virus G Protein-Pseudotyped Lentiviral Vector Preparations Carry DNA and Stimulate Antiviral Responses via Toll-Like Receptor 9{triangledown}

Andreas Pichlmair,1 Sandra S. Diebold,1,{dagger} Stephen Gschmeissner,2 Yasuhiro Takeuchi,3 Yasuhiro Ikeda,3,{ddagger} Mary K. Collins,3 and Caetano Reis e Sousa1*

Immunobiology Laboratory,1 Electron Microscopy Unit, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom,2 Infection and Immunity, University College London, Windeyer Building, 46 Cleveland Street, London W1T 4JF, United Kingdom3

Received 21 August 2006/ Accepted 20 October 2006

Recombinant lentiviral vectors (LVs) are commonly used as research tools and are being tested in the clinic as delivery agents for gene therapy. Here, we show that Vesicular stomatitis virus G protein (VSV-G)-pseudotyped LV preparations produced by transient transfection are heavily contaminated with tubulovesicular structures (TVS) of cellular origin, which carry nucleic acids, including the DNA plasmids originally used for LV generation. The DNA carried by TVS can act as a stimulus for innate antiviral responses, triggering Toll-like receptor 9 and inducing alpha/beta interferon production by plasmacytoid dendritic cells (pDC). Removal of TVS markedly reduces the ability of VSV-G-pseudotyped LV preparations to activate pDC. Conversely, virus-free TVS are sufficient to stimulate pDC and act as potent adjuvants in vivo, eliciting T- and B-cell responses to coadministered proteins. These results highlight the role of by-products of virus production in determining the immunostimulatory properties of recombinant virus preparations and suggest possible strategies for diminishing responses to LVs in gene therapy and in research use.

* Corresponding author. Mailing address: Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Phone: 44 20 7269 2832. Fax: 44 20 7269 2833. E-mail: caetano{at}cancer.org.uk.

{triangledown} Published ahead of print on 1 November 2006.

{dagger} Present address: King's College London, Guy's, King's and St. Thomas Medical School, Peter Gorer Department of Immunobiology, Division of Immunology, Infection and Inflammatory Diseases, 2nd Floor New Guy's House, Guy's Hospital, London Bridge, London SE1 9RT, United Kingdom.

{ddagger} Present address: Molecular Medicine Program, Guggenheim Building 18-11c, Mayo Clinic College of Medicine, 200 First Street S.W., Rochester, MN 55905.

Journal of Virology, January 2007, p. 539-547, Vol. 81, No. 2
0022-538X/07/$08.00+0     doi:10.1128/JVI.01818-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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