Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways

Abstract

Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.

FIG. 1.

Induction of mainly type I IFNs upon adenoviral infection. (A) GM-CSF DCs were generated from C57BL/6 bone marrow cells in the presence of GM-CSF and IL-4 for 5 days. On day 5, DCs (1 × 10⁶ cells/ml) were either unstimulated (medium) or stimulated with PGN, LPS, CpG ODN (CpG), Ad-lacZ (Ad) or UV-inactivated Ad-lacZ (UV-Ad) for 18 h. Culture supernatants were analyzed by ELISA for secretion of IL-6, IL-12, IFN-α, and IFN-β. (B) Mice were administered intravenously with Ad-lacZ (Ad) or LPS. Serum samples were harvested 6 h later and analyzed for secretion of IL-6 and IFN-α.

FIG. 2.

Production of type I IFNs by pDCs versus non-pDCs in response to adenoviral infection. (A) Isolation of splenic pDCs and cDCs. Splenocytes from C57BL/6 mice were enriched for CD11c⁺ DCs by a bovine serum albumin gradient and microbeads, and the CD11c⁺ DC-enriched fraction was then stained with anti-CD11c and anti-B220 and sorted by FACS into pDCs and cDCs. Aliquots of sorted cells (postsort) were analyzed by FACS to determine the purity of pDCs (CD11c⁺ B220⁺ mPDCA-1⁺) and cDCs (CD11c⁺ B220⁻ mPDCA-1⁻). (B) Isolation of hepatic Kupffer cells. Kupffer cells were enriched from mouse livers after collagenase perfusion and gradient centrifugation. The Kupffer cell-enriched fraction was then stained with antibody to F4/80, a Kupffer cell marker, and subjected to FACS sorting. The FACS plot indicates the purity of Kupffer cells after sorting. (C) Splenic pDCs, cDCs, hepatic Kupffer cells (KC), or peritoneal macrophages (MΦ) at 2.5 × 10⁵ cells/ml were either unstimulated (medium) or stimulated with Ad-lacZ (Ad) at an MOI of 250 for 18 h, and the supernatants were measured for IFN-α and IFN-β. (D) Transduction of pDCs and cDCs by adenovirus. Purified pDCs and cDCs were infected with Ad-GFP or Ad-lacZ at an MOI of 250, and 24 h later cells were analyzed for GFP expression by FACS. The percentage of GFP⁺ cells is indicated.

FIG. 3.

Innate immune recognition of adenovirus by pDCs is mediated by TLR9 and dependent on MyD88. (A) Splenic pDCs purified from WT, MyD88^−/−, or TRIF^−/− C57BL/6 mice were stimulated with Ad-lacZ at an MOI of 250 for 18 h at 2.5 × 10⁵ cells/ml and assayed for the secretion of IFN-α. (B) Splenic pDCs purified from WT or TLR9^−/− C57BL/6 mice were stimulated with Ad-lacZ at an MOI of 250 or CpG ODN (CpG) for 18 h at 2.5 × 10⁵ cells/ml and assayed for the secretion of IFN-α. (C) pDCs (5 × 10⁵ cells/ml) generated from bone marrow cells of WT, MyD88^−/−, TRIF^−/−, or TLR9^−/− C57BL/6 mice in the presence of Flt-3L were stimulated with Ad-lacZ at an MOI of 250 for 18 h and assayed for the secretion of IFN-α. (D) WT, MyD88^−/−, or TLR9^−/− mice were administered Ad-lacZ intravenously. Serum samples were harvested from these mice 12 h later and analyzed for secretion of IFN-α. Serum from uninfected naïve mice was used as a control.

FIG. 4.

Innate immune recognition of adenovirus by non-pDCs is TLR independent. (A to C) Splenic cDCs (A), hepatic Kupffer cells (B), and peritoneal macrophages (C) purified from WT, MyD88^−/−, TRIF^−/−, or TLR9^−/− mice were stimulated with Ad-lacZ at an MOI of 250 for 18 h at 2.5 × 10⁵ cells/ml and assayed for secretion of IFN-α. (D) GM-CSF DCs generated from bone marrow cells of WT, MyD88^−/−, TRIF^−/−, or TLR9^−/− mice in the presence of GM-CSF and IL-4 were stimulated with Ad-lacZ at an MOI of 250 for 18 h at 1 × 10⁶ cells/ml and assayed for secretion of IFN-α.

FIG. 5.

Cytosolic recognition of adenoviral DNA triggers a TLR-independent pathway. (A) GM-CSF DCs at 1 × 10⁶ cells/ml were incubated with “naked” adenoviral DNA (Ad-DNA only), an adenoviral DNA-liposome mix (Ad-DNA/liposome), or a mouse genomic DNA-liposome mix (gDNA/liposome) at a final DNA concentration of 0, 0.2, 1, or 5 μg/ml for 18 h. The supernatants were measured for IFN-α and IL-6 by ELISA. (B) GM-CSF DCs generated from WT, MyD88^−/−, TRIF^−/−, or TLR9^−/− mice were either mock transfected or transfected with 5 μg/ml of adenoviral DNA/liposome mix (Ad DNA) for 18 h and assayed for IFN-α and IL-6.

FIG. 6.

Critical role of type I IFNs in innate immune responses to adenoviral vectors. Ad-lacZ was injected intravenously into WT or IFN-αβR^−/− (IFN-αβR^−/−) 129/Sv mice. (A) Six hours later, sera were measured for IL-6 and IL-12. (B) Two or 72 h later, total genomic DNA was isolated from the liver and analyzed for adenoviral genome copies by quantitative real-time PCR. Data represent adenoviral copies per μg of total genomic DNA.

FIG. 7.

Critical role of type I IFNs in adaptive immune responses to adenoviral vectors. Ad-lacZ was injected intravenously into WT or IFN-αβR^−/− 129/Sv mice. (A) Splenocytes from WT or IFN-αβR^−/− mice at day 10, along with uninfected WT splenocytes (Control) were restimulated with Ad-lacZ at an MOI of 1, 10, or 100 for 72 h. Proliferation of virus-specific T cells was analyzed by [³H]thymidine incorporation. Data reflect the mean ± SD of the stimulation index, calculated by dividing ³H counts in cpm in the presence of viral stimulation by those in the absence of stimulation, as a function of different virus doses. (B) Splenocytes from infected WT or IFN-αβR^−/− mice at day 10, or uninfected splenocytes, were restimulated in vitro for 5 days and assayed for virus-specific CTL using a standard ⁵¹Cr release method. Data represent the percentage of specific lysis as a function of different effector-to-target ratios. (C) At 3, 10, or 21 days after infusion of virus, liver tissues were harvested and cryosections were stained for LacZ expression by X-Gal histochemistry. On day 10, paraffin sections were also prepared and stained with hematoxylin and eosin for histopathology. (D) Serum samples were harvested from WT or IFN-αβR^−/− mice 21 days after infection (Ad), or from uninfected mice (Control), for the measurement of neutralizing antibody (Ab) titers to adenoviral vectors.

FIG. 8.

Type I IFN blockade diminishes innate and adaptive immune responses to adenovirus. Ad-lacZ (2 × 10⁹ PFU) was injected intravenously into C57BL/6 mice that had been treated with neutralizing antibodies to IFN-α and IFN-β (IFN-αβ Ab) or a control antibody (Control Ab). (A) At 3 days after administration of virus, liver tissues were harvested, and total genomic DNA was isolated and analyzed for adenoviral genome copies by quantitative real-time PCR. Data represent adenoviral copies per μg of total murine liver genomic DNA. (B) Ten days later, splenocytes were restimulated with Ad-lacZ at an MOI of 1, 10, or 100. Proliferation of virus-specific T cells was analyzed by [³H]thymidine incorporation. Data reflect the mean ± SD of the stimulation index as described in legends to Fig. 7. (C) Ten days after infusion of virus, liver tissues were harvested. Cryosections were stained for LacZ expression by X-Gal histochemistry, and paraffin sections were stained with H&E for histopathology.