Complement is an essential component of the immune response to adeno-associated virus vectors

Abstract

Adeno-associated virus (AAV) vectors are associated with relatively mild host immune responses in vivo. Although AAV induces very weak innate immune responses, neutralizing antibodies against the vector capsid and transgene still occur. To understand further the basis of the antiviral immune response to AAV vectors, studies were performed to characterize AAV interactions with macrophages. Primary mouse macrophages and human THP-1 cells transduced in vitro using an AAV serotype 2 (AAV2) vector encoding green fluorescent protein did not result in measurable transgene expression. An assessment of internalized vector genomes showed that AAV2 vector uptake was enhanced in the presence of normal but not heat-inactivated or C3-depleted mouse/human serum. Enhanced uptake in the presence of serum coincided with increased macrophage activation as determined by the expression of NF-kappaB-dependent genes such as macrophage inflammatory protein 2 (MIP-2), interleukin-1beta (IL-1beta), IL-8, and MIP-1beta. AAV vector serotypes 1 and 8 also activated human and mouse macrophages in a serum-dependent manner. Immunoprecipitation studies demonstrated the binding of iC3b complement protein to the AAV2 capsid in human serum. AAV2 did not activate the alternative pathway of the complement cascade and lacked cofactor activity for factor I-mediated degradation of C3b to iC3b. Instead, our results suggest that the AAV capsid also binds complement regulatory protein factor H. In vivo, complement receptor 1/2- and C3-deficient mice displayed impaired humoral immunity against AAV2 vectors, with a delay in antibody development and significantly lower neutralizing antibody titers. These results show that the complement system is an essential component of the host immune response to AAV.

FIG. 1.

Transduction of macrophage lines and primary macrophages by AAV and adenovirus vectors. Transduction of human muscle TE671 cells, primary mouse bone marrow macrophages (pr. MO), the human monocytic cell line THP-1, or differentiated THP-1 cells (diff. THP-1) with an AAV2 vector (1 × 10⁴ part/cell) or an adenovirus vector encoding GFP (1 × 10³ part/cell). In contrast to Ad-GFP, AAV-GFP does not efficiently transduce macrophage lines or primary macrophages and does not induce any phenotypic change (insets). Data are representative samples of at least four independent vector transductions.

FIG. 2.

AAV vector genome uptake in macrophages in vitro. (A) Southern blot analysis of intracellular vector genomes, blotting for the GFP transgene, 6 h after transduction of primary mouse bone marrow macrophages with AAV2-GFP. Cells were transduced in the presence of normal tissue culture medium containing 10% FBS, serum-free medium (S-free), medium containing 10% MP, or medium containing 10% HI-MP. Vector genome uptake is significantly higher in the presence of MP than in the presence of normal tissue culture medium (FBS), S-free, or HI-MP (means ± standard deviations [SD]) (**, P < 0.01; ***, P < 0.001; n = 3). Differences between FBS, S-free, and HI-MP were not significant (n = 3). (B) Southern blot showing internalized AAV vector genomes in differentiated THP-1 cells blotting for the GFP transgene. Cells were transduced in tissue culture medium supplemented with 10% FBS, 10% HS, or 10% HS-C3. DNA quantities were determined by phosphorimaging (ss, single-stranded vector genome; ds, double-stranded monomer). Vector genome uptake is significantly higher in the presence of HS than in the presence of normal tissue culture medium (FBS) or HS-C3 (means ± SD) (*, P < 0.05; n = 3). Differences between FBS and HS-C3 were not significant (n = 3). VH, vehicle.

FIG. 3.

AAV vector-induced chemokine and cytokine expression in macrophages in vitro. (A) MIP-2 RNA expression 90 min after AAV2-GFP transduction (1 × 10⁵ part/cell) of primary mouse bone marrow macrophages. Cells were transduced in the presence of normal tissue culture medium containing 10% FBS, serum-free medium (S-free), medium containing 10% MP, or medium containing 10% HI-MP. Quantification of MIP-2 expression is based on phosphorimaging of RNase protection assays and normalized against GAPDH. AAV induced the expression of MIP-2 significantly above baseline (vehicle [VH]) under all serum conditions (means ± standard deviations [SD]) (***, P < 0.001). Furthermore, AAV-induced MIP-2 expression is significantly higher in the presence of MP than in the presence of normal tissue culture medium (FBS), S-free, or HI-MP (means ± SD) (***, P < 0.001; n = 3). (B and C) RNase protection assay of differentiated THP-1 cell RNA 90 min after transduction with AAV2-GFP (1 × 10⁵ part/cell). Quantification of chemokine/cytokine expression is based on phosphorimaging of RNA blots and normalized against GAPDH. Cells were transduced in tissue culture medium supplemented with 10% FBS, 10% HS, or 10% HI-HS. Cytokine/chemokine gene expression in the presence of HS was significantly increased compared to that in the presence of FBS (means ± SD) (*, P < 0.05 for MIP-1β; **, P < 0.01 for IL-8; ***, P < 0.001 for IL-1β; n = 3) or HI-HS (means ± SD) (**, P < 0.01 for MIP-1β; ***, P < 0.001 for IL-8 and IL-1β; n = 3).

FIG. 4.

Chemokine and cytokine expression in macrophages transduced with AAV1 and AAV8 in vitro. (A) RNase protection assay of THP-1 cell RNA 90 min after transduction with AAV1-GFP (1 × 10⁵ part/cell) or AAV8-GFP (1 × 10³, 1 × 10⁴, or 1 × 10⁵ part/cell). Quantification of chemokine expression following transduction with AAV8-GFP (1 × 10⁵ part/cell) or AAV1-GFP (1 × 10⁵ part/cell) by phosphorimaging normalized against GAPDH. (B) RNase protection assay of primary mouse bone marrow macrophage RNA 90 min after transduction with AAV8-GFP (1 × 10⁵ part/cell). Quantification of MIP-2 and IP-10 expression by phosphorimaging normalized against GAPDH. Cells were transduced in medium supplemented with either 10% MP or 10% HI-MP. In the presence of MP, AAV8-GFP induced the expression of MIP-2 and IP-10 RNA significantly above baseline (means ± standard deviations) (***, P < 0.001; n = 3). Furthermore, AAV-induced MIP-2 and IP-10 expression was significantly increased in the presence of MP compared to that in the presence of HI-MP (***, P < 0.001; n = 3). VH, vehicle.

FIG. 5.

AAV interaction with complement. (A) Pooled HS contains neutralizing anti-AAV IgG antibodies. Transduction of HEK 293 cells with AAV2-GFP in the presence of tissue culture medium supplemented with 10% FBS, 10% pooled HS, or 10% HS-IgG. Pooled HS abrogates GFP transgene expression. (B) Southern blot of internalized AAV vector genomes blotting for the GFP transgene. HEK 293 cells were transduced with AAV2-GFP, and total DNA was harvested and analyzed for AAV vector genomes at 6 h. Pooled HS abrogates viral vector entry into HEK 293 cells. Representative Southern blot of at least three independent experiments. VH, vehicle; ss, single-stranded vector genome; ds, double-stranded monomer. (C) Diagram of C3 structure. C3 is composed of two chains, α and β. Upon activation C3a is released, leaving C3b, consisting of the truncated α′ chain (107 kDa) and the β chain (75 kDa). Factor I and a cofactor cleave the α′ chain at two locations, thereby creating iC3b, which consists of β, α1, and α2 chains (75, 67, and 40 kDa, respectively). (D) Viral complement coimmunoprecipitation. AAV2-GFP, Ad-GFP, and HSV-1 were immunoprecipitated from HS and analyzed by immunoblotting C3 complement components or AAV capsid proteins. Staining for human IgG heavy (H-chain) and light (L-chain) chains served as the loading control. Representative blot from three independent experiments. (E) Coimmunoprecipitation of AAV and complement in the absence of HS. AAV was incubated with complement component C3, C3b, or C3b mixed with factor H (fH) and factor I to generate iC3b, followed by immunoprecipitation of the complement components with a polyclonal anti-C3 antibody. Precipitate (IP) and starting material (ext) were analyzed by immunoblotting (IB) for AAV capsid proteins, C3 complement components, and fH.

FIG. 6.

AAV-induced complement activation. (A) Complement activation in HS after stimulation with adenovirus or AAV vectors in the presence or absence of neutralizing antibodies. Values above bars represent the concentrations of vector particles per milliliter of HS. Vehicle (VH) or vector was incubated with HS at the indicated concentration for 90 min at 37°C. C3a-desArg levels were determined by ELISA. EGTA, HS pretreated with 1/10 volume of 0.1 M EGTA to inhibit the classical pathway of complement activation. Values represent mean C3a concentrations ± standard deviations (n = 3). In IgG-depleted or EGTA-treated HS, AAV and adenovirus vectors did not generate a significant increase of C3a above baseline (NS, P > 0.05). In pooled HS, adenovirus vectors increased C3a significantly over baseline (**, P < 0.01). At 1 × 10¹¹ part/ml, AAV did not generate a significant increase of C3a above baseline (NS, P > 0.05). (B) Complement activation in HS after stimulation with high titers of adenovirus or AAV vectors. At 1 × 10¹² part/ml, AAV induced a measurable increase of C3a over baseline but less than the equivalent titer of adenovirus. (C) Analysis of factor I cofactor activity of AAV for C3b. C3b (200 ng) was incubated with 1 μg of factor I and AAV (4 × 10¹⁰ particles), adenovirus vectors (4 × 10¹⁰ particles), or HSV-1 (4 × 10⁸ particles) for 1 h. C3b cleavage was analyzed by Western blotting. Reaction mixes without virus vectors and with or without fI and factor H (fH) were included as controls. Cofactor activity is demonstrated by the appearance of the α′ chain cleavage fragments at 67 and 40 kDa. HSV-1, but not AAV or adenovirus, has cofactor activity. (D) Coimmunoprecipitation of AAV and human fH using anti-human fH antibody. Starting material (ext) and immunoprecipitates (IP) were immunoblotted (IB) for AAV capsid proteins and fH. NS, not significant; **, P < 0.01.

FIG. 7.

Innate immune response to AAV in C3^−/− mice. RNase protection assay of mouse liver RNA from C3^−/− mice or wild-type (WT) C57BL/6 mice 1 h following an intravenous injection of AAV2-GFP (2.5 × 10¹¹ particles). Mice either were naïve (N) or had been immunized (I) 2 weeks earlier with AAV2-GFP. Quantification of IP-10 and MIP-1β expression is based on phosphorimaging of RNA blots and normalized against GAPDH. AAV-induced chemokine and cytokine mRNA expression in C3^−/− mice is not significantly different from that in wild-type mice (means ± standard deviations). VH, vehicle.

FIG. 8.

Humoral immune response to AAV vectors in CR1/2^−/− and C3^−/− mice. (A) AAV2-GFP transduction of HEK 293 cells and Ad-GFP transduction of TE671 cells in the presence of tissue culture medium supplemented with 5% naïve mouse serum or 5% serum from AAV2-GFP-immunized C57BL/6 wild-type (WT) or CR1/2^−/− mice. Serum from immunized WT but not CR1/2^−/− mice abrogates AAV transgene expression in HEK 293 cells. Adenovirus transgene expression is abrogated by serum from both WT and CR1/2^−/− mice. Representative images of at least three independent transductions are shown. (B) AAV and adenovirus neutralizing antibody titer development over time in WT and CR1/2^−/− mice following AAV2-GFP administration. The antibody titer is displayed as the level that inhibited transgene expression 50% in tissue culture as determined by serial dilutions. Neutralizing antibody titers were significantly higher for AAV2-GFP-injected WT mice than for CR1/2^−/− mice at 2 weeks (means ± standard deviations [SD]) (**, P < 0.01; n = 3). Ad-GFP-injected WT and CR1/2^−/− animals displayed no significant difference in neutralizing antibody titer at 2 weeks. (C) AAV neutralizing antibody titer in WT and C3^−/− mice receiving AAV2-GFP. Neutralizing antibody titers were significantly higher for WT mice than for C3^−/− mice at 1, 2, and 3 weeks (means ± SD) (*, P < 0.05; **, P < 0.01; n = 3 to 5).