Tubulovesicular structures within vesicular stomatitis virus G protein-pseudotyped lentiviral vector preparations carry DNA and stimulate antiviral responses via Toll-like receptor 9.
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.
Pichlmair
Andreas
A
Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
Diebold
Sandra S
SS
Gschmeissner
Stephen
S
Takeuchi
Yasuhiro
Y
Ikeda
Yasuhiro
Y
Collins
Mary K
MK
Reis e Sousa
Caetano
C
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000276:immunology*; D003713:Dendritic Cells / Q000276:immunology*; D005822:Genetic Vectors; D006801:Humans; D007114:Immunization; D016898:Interferon-alpha / Q000096:biosynthesis; D016086:Lentivirus / Q000235:genetics / Q000276:immunology* / Q000648:ultrastructure*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D011995:Recombination, Genetic; D051217:Toll-Like Receptor 9 / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology
C028391:G protein, vesicular stomatitis virus; D016898:Interferon-alpha; D008562:Membrane Glycoproteins; D051217:Toll-Like Receptor 9; D014759:Viral Envelope Proteins; D004247:DNA