Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells

Abstract

The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.

FIG. 1.

Transduction of mouse muscle by AAV serotype vectors. AAV LacZ-expressing vectors (10¹⁰ vp) were injected intramuscularly into five mice/group. β-Galactosidase activity in the muscle was examined 2 weeks later using the Beta-Glo Assay System. An asterisk indicates a significant difference between the two groups (P < 0.05). RLU, relative light units.

FIG. 2.

Transduction of mouse purified DCs by AAV serotype vectors. (a) Mouse CD11c⁺ DCs were transduced with LacZ-expressing AAV vectors at 10⁴ vp/cell (triplicate for each sample). Two days after infection, the cells were stained with X-Gal (upper panel), and the β-galactosidase activity was measured using the Beta-Glo Assay System (bottom panel). The data presented were averaged from three separate experiments. The asterisk indicates a significant difference when AAV2-LacZ-transduced cells were compared to AAV1-LacZ-, AAV3-LacZ-, AAV4-LacZ-, AAV7-LacZ-, and AAV8-LacZ-transduced cells and mock-transduced cells; the double asterisk indicates a significant difference when AAV5-LacZ-transduced cells were compared to AAV2-LacZ-transduced cells. RLU, relative light units. (b) Mouse CD11c⁺ DCs were transduced with AAV-LacZ vectors at 10³ to 10⁵ vp/cell (triplicate for each sample). Two days after transduction, the cells were stained with X-Gal, and the percentages of LacZ-expressing DCs (FDG⁺ CD11c⁺ DCs) were determined. The data presented were averaged from three separate experiments. (c) Enriched immature DCs were transduced with 10⁵ vp/cell of various AAV-LacZ vectors (triplicate for each sample). Two days after transduction, the cells were treated with PE-conjugated anti-mouse CD11c Ab and FDG followed by flow cytometric analysis. The data provided represent the fraction of FDG/CD11c dual-positive cells as a percentage of the total population of CD11c⁺ cells. The data presented were averaged from three independent experiments.

FIG. 2.

Transduction of mouse purified DCs by AAV serotype vectors. (a) Mouse CD11c⁺ DCs were transduced with LacZ-expressing AAV vectors at 10⁴ vp/cell (triplicate for each sample). Two days after infection, the cells were stained with X-Gal (upper panel), and the β-galactosidase activity was measured using the Beta-Glo Assay System (bottom panel). The data presented were averaged from three separate experiments. The asterisk indicates a significant difference when AAV2-LacZ-transduced cells were compared to AAV1-LacZ-, AAV3-LacZ-, AAV4-LacZ-, AAV7-LacZ-, and AAV8-LacZ-transduced cells and mock-transduced cells; the double asterisk indicates a significant difference when AAV5-LacZ-transduced cells were compared to AAV2-LacZ-transduced cells. RLU, relative light units. (b) Mouse CD11c⁺ DCs were transduced with AAV-LacZ vectors at 10³ to 10⁵ vp/cell (triplicate for each sample). Two days after transduction, the cells were stained with X-Gal, and the percentages of LacZ-expressing DCs (FDG⁺ CD11c⁺ DCs) were determined. The data presented were averaged from three separate experiments. (c) Enriched immature DCs were transduced with 10⁵ vp/cell of various AAV-LacZ vectors (triplicate for each sample). Two days after transduction, the cells were treated with PE-conjugated anti-mouse CD11c Ab and FDG followed by flow cytometric analysis. The data provided represent the fraction of FDG/CD11c dual-positive cells as a percentage of the total population of CD11c⁺ cells. The data presented were averaged from three independent experiments.

FIG. 3.

Transduction of mouse unpurified bone marrow cells and splenocytes by AAV serotype vectors. Mouse bone marrow cells and splenocytes were transduced with 10⁵ vp/cell of AAV-LacZ vectors. Two days after transduction, the cells were treated with FDG and the splenocytes were stained with anti-mouse CD3 (T cells), anti-mouse CD11b (macrophages), or anti-mouse CD19 (B cells) antibody followed by flow cytometric analysis. Panels show results from one of three independent experiments.

FIG. 4.

Transduction of enriched human DCs and unpurified PBMCs by AAV serotype vectors. Human enriched DCs or PBMCs were transduced with 10⁵ vp/cell of AAV-LacZ vectors. Two days after transduction, the DCs were treated with FDG and anti-human CD11c Ab, and PBMCs were treated with FDG and anti-human CD3 (T cells), anti-human CD11b (macrophages), or anti-human CD19 (B cells) antibody followed by flow cytometric analysis. Panels show results from one of three independent experiments.

FIG. 5.

Expression of HIV gp160 and HIV-specific serum IgG titer by AAV serotype vectors. (a) HEK293 cells were transduced with AAV vectors carrying the HIV Env gp160 gene. Two days after infection, cell lysates were analyzed by Western blotting using anti-HIV Env MAb and anti-human β-actin MAb. (b) BALB/c mice (five mice/group) were immunized with AAV-HIV vectors on days 0, 14, and 28. HIV-specific serum IgG was measured 1 month after the final immunization. The BacAAV vectors were generated in insect cells, whereas other vectors were produced in HEK293 cells. The asterisks indicate significant differences between the two groups (P < 0.05).

FIG. 6.

HIV-specific cell-mediated immune responses induced by AAV vector vaccination. BALB/c mice (five mice/group) were immunized with AAV-HIV vectors on days 0, 14, and 28. Five mice per group for the IFN-γ ELISPOT and tetramer assay and five mice per group for the in vivo CTL assay were used. The HIV-specific IFN-γ ELISPOT assay was performed (a), the percentage of HIV-specific tetramer binding CD8⁺ cells was determined (b), and the in vivo CTL assay was performed (c) 2 weeks after the final immunization. BacAAV vectors were generated in insect cells, whereas other AAV vectors were generated in HEK293 cells. Asterisks indicate significant differences between the two groups (P < 0.05). Panels b and c show results from individual representative animals, while the data represent the averages of five mice/group.

FIG. 7.

Immune responses induced by ex vivo DC transduction. Mouse enriched DCs were transduced with 10⁵ vp/cell of AAV-HIV vectors for 2 days. The cells were stained with PE-conjugated anti-mouse CD11c antibody plus mouse anti-HIV gp120 antibody followed by staining with FITC-conjugated anti-mouse IgG. The HIV gp160 expression of DC surface was analyzed by flow cytometry (a). Enriched mouse DCs were transduced with AAV-HIV vectors for 2 h, washed with PBS (−), and injected intravenously into recipient BALB/c mice (five mice/group). HIV-specific responses were detected using the tetramer assay 1 week after administration. Dot plots show results from individual representative animals, while the data shown in the upper right quadrant represent the averages of five mice/group (b). Neg. Con., negative control.