Tubulovesicular structures within vesicular stomatitis virus G protein-pseudotyped lentiviral vector preparations carry DNA and stimulate antiviral responses via Toll-like receptor 9

Abstract

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.

FIG. 1.

LV preparations induce IFN-α production by pDC. (A) IFN-α secretion by BM cells after treatment with preparations of VSV-G-pseudotyped LVs at the indicated multiplicity of infections (MOIs) or with CpG (0.5 μg/ml; positive control). Supernatant (SN) of untransfected cells was concentrated as for the LV preparation and used as a negative control. (B) Intracellular staining for IFN-α in BM cells stimulated with LVs at an MOI of 0.2 for 6 h. Left panel, IFN-α versus CD11c. Right panel, Ly6C and B220 expression on IFN-α-positive cells gated as indicated in the left panel. Numbers represent the percentages of cells in each gate or quadrant. (C) IFN-α secretion by B220⁺ and B220⁻ fractions of BM cells taken from RAG-2^−/− mice after treatment with VSV-G-pseudotyped LVs (MOI of 0.2) or CpG. The B220⁺ fraction consisted of >90% pDC; the B220⁻ fraction contained around 7% pDC (not shown). n.d., not detected. Error bars indicate standard deviations of triplicates.

FIG. 2.

VSV-G expression is required for the IFN-α response to LVs. (A) BM cells were stimulated with LV preparations (multiplicity of infection of 1) pseudotyped with the indicated glycoproteins; IFN-α was measured after overnight culture. (B) Stimulation of BM with concentrated supernatants from 293FT cells transfected with the indicated combinations of plasmids to generate preparations of VSV-G-pseudotyped LV, LV lacking viral RNA (LVΔvRNA), or LV lacking the envelope protein (LVΔenv). Concentrated supernatants from untransfected cells (SN) or from cells transfected with plasmids encoding Rev plus VSV-G (VSV-G) or Rev plus viral RNA (vRNA) were tested as controls. Where applicable, stimuli were normalized for HIV p24 content (data not shown). (C) Concentrated supernatant from VSV-G-transfected cells (VSV-G-SN) or CpG was preincubated with a neutralizing anti-VSV-G or control MAb before being added to BM cells. IFN-α in supernatants was measured 16 h later. n.d., not detected. Error bars indicate standard deviations of triplicates.

FIG. 3.

VSV-G transfection induces formation of TVS that carry cellular proteins and constitute the main pDC stimulus in VSV-G-pseudotyped LV preparations. (A) Electron micrographs of supernatants (left column) and transfected cells (right column) producing VSV-G-pseudotyped LV, VSV-G only, or LV without envelope protein (LVΔenv). Bars, 200 nm (left column) and 1 μm (right column). Black arrows indicate TVS; white arrows show virus particles. *, cell cytoplasm. (B) Aliquots of preparations of LVs, TVS, and LVΔenv were blotted for the indicated proteins. (C) VSV-G-LV preparations were fractionated on a continuous sucrose gradient. Each fraction was assessed for IFN-α induction in BM cells and for virus titer (GFU) on 293T cells. Western blots show the amounts of VSV-G and HIV p24 in each fraction. Fraction 8 represents the bottom of the gradient (dense fraction).

FIG. 4.

IFN-α induction by LV preparations and TVS is dependent on TLR9 signaling. (A) BM cells from C57BL/6 or MyD88^−/− mice were cultured in medium alone (no stim), with LVs (multiplicity of infection of 0.1) or were electroporated with poly(I:C) (0.5 μg) as described previously (15). IFN-α was measured after overnight incubation. (B) IFN-α from TLR7^−/− or wild-type BM cells after stimulation with LVs (multiplicity of infection of 0.1), R848 (1 μg/ml), or CpG (0.5 μg/ml). (C) BM cells from wild type or TLR9^−/− mice were stimulated with LVs (multiplicities of infection of 0.1 and 0.01), VSV-G TVS (10× and 1× concentrated), Loxoribine (20 mM), or CpG. IFN-α was measured after overnight culture. n.d., not detected. Error bars indicate standard deviations of triplicates.

FIG. 5.

TVS and LV preparations generated by transient transfection contain plasmid DNA. (A) RT-PCR and PCR on RNA and DNA isolated from preparations of LVs, LVs lacking the envelope protein (LVΔenv), or TVS (concentrated supernatant from VSV-G-transfected cells). (B) DNA extracted from LV preparations was used to transform bacteria. Ampicillin-resistant individual colonies were picked, and the plasmid DNA extracted and analyzed by digestion with restriction enzymes. The graph shows the frequency of bacterial colonies containing each plasmid (average ± standard deviation from three experiments; n > 150 colonies). (C) LV preparations generated by retroviral transduction do not stimulate IFN-α production by BM cells. VSV-G was introduced into STAR-HV cells by transient transfection or by retroviral transduction. Supernatant containing VSV-G-pseudotyped LV (multiplicity of infection of 0.1) was added to BM cells. IFN-α was measured by ELISA 16 h after treatment. CpG (0.5 μg/ml) was used as a positive control. n.d., not detected.

FIG. 6.

TVS act as adjuvants to induce adaptive immune responses. C57BL/6 mice were immunized intraperitoneally with egg white in PBS (n = 2) or with added TVS (n = 4) or poly(I:C) (PIC) (n = 2). (A) Contour plots show OVA/H-2K^b tetramer-positive Thy1.2⁺ cells in blood of representative mice 1 week after immunization. The graph shows the average (±standard deviation) frequency of OVA/H2-K^b tetramer-positive Thy1.2⁺ cells for all mice. *, P < 0.05; (as determined by Student's t test, compared to PBS-OVA-immunized mice). (B) At 10 days after immunization, mice were challenged with congenic CD45.1 splenocytes loaded with 20 nM (carboxyfluorescein [CFSE] low), 200 nM (CFSE intermediate), or 0 nM (CFSE high) of OVA peptide (SIINFEKL). Histograms show target cells (gated on CD45.1) from representative mice at 48 h after injection. The graph shows the amount of specific killing (average ± standard deviation) in all groups from one representative experiment. *, P < 0.05; **, P < 0.001 (as determined by Student's t test, compared to PBS-OVA-immunized mice). n.d., not detected. (C) At 12 days after immunization, splenocytes were isolated, cultured overnight in the absence or presence of FCS, and stained for intracellular IFN-γ. Contour plots show gated Thy1.2⁺ cells from representative mice. Values in the bottom right panel ranged from 0.16 to 2.2% IFN-γ⁺ Thy1.2⁺ CD4⁺ cells. (D) At 12 days after immunization, sera of mice were tested for the presence of specific antibodies against OVA and FCS. Data are displayed as titration curves from individual representative mice. IgG, immunoglobulin G; OD, optical density.