Distinct immune responses to transgene products from rAAV1 and rAAV8 vectors - PubMed (original) (raw)

Distinct immune responses to transgene products from rAAV1 and rAAV8 vectors

Yuanqing Lu et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Recently developed serotypes of recombinant adeno-associated virus (rAAV) vectors have significantly enhanced the use of rAAV vectors for gene therapy. However, host immune responses to the transgene products from different serotypes remain uncharacterized. In the present study, we evaluated the differential immune responses to the transgene products from rAAV1 and rAAV8 vectors. In non-obese diabetic (NOD) mice, which have a hypersensitive immunity, rAAV serotype 1 vector (rAAV1-hAAT) induced high levels of both humoral and cellular responses, while rAAV8-hAAT did not. In vitro studies showed that rAAV1, but not rAAV8 vector transduced dendritic cells (DCs) efficiently. In vivo studies indicated that vector transduction of DCs was essential for the immune responses; while the presence of a transgene product (or foreign gene product produced by host cells) was not immunogenic. Intriguingly, preimmunization with rAAV8-hAAT vector or with serum of hAAT transgenic NOD mouse induced immune tolerance to rAAV1-hAAT injection. These results demonstrate the immunogenic differences of rAAV1 and rAAV8 and imply tremendous potential for these vectors in different applications, where an immune response to transgene is to be either elicited or avoided.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Human AAT expression and immune responses to hAAT in NOD mice injected with rAAV1-hAAT or rAAV8-hAAT. AAV1-CB-hAAT, AAV8-CB-hAAT, and AAV1-CB-GFP (2 × 1011 gv/mouse) were injected into 4-week-old NOD mice by IP injection or IM injection. Serum hAAT and anti-hAAT antibody levels were detected by ELISA. Cellular immune response was measured by IFN-γ ELISpot assay. (A) Serum hAAT levels in NOD mice (n = 10 in each group). Each line represents the average levels of each group. Filed diamond (AAV8 ip): IP injected with rAAV8-CB-hAAT; open diamond (AAV8 im): IM injected with rAAV8-CB-hAAT; filed triangle (AAV1ip): IP injected with rAAV1-CB-hAAT; open triangle (AAV1 im): IM injected with rAAV1-CB-hAAT. (B) IFN-γ ELISPOT assay using splenocytes from treatment and control groups (n = 3 in each group). The number of spot-forming cells (SFC) per 105 spleen cells is shown in the y axis. (C–F) Anti-hAAT antibody levels in rAAV1-hAAT (IM)-injected group (C), in rAAV1-hAAT (IP)-injected group (D), rAAV8-hAAT (IM)-injection group (E) and in rAAV8-hAAT (IP)-injection group (F). In C–F, each line shows the antibody levels in an individual animal (n = 10).

Fig. 2.

Fig. 2.

Transduction and transplantation of dendritic cells. (A) Detection of rAAV vector DNA in bone marrow-derived DCs after in vitro infection with AAV1-hAAT or AAV8-hAAT or saline (MOI = 2.8 × 104 vg/cell) by real-time PCR. Each bar shows the average concentration of hAAT DNA (molecules per DCs) in each group (n = 4). (B–D) Transgene expression from DCs (B), mouse macrophage cell line, RAW 264.7 (C), and mouse muscle cell line C2C12 (D) after infection with AAV1-hAAT or AAV8- hAAT or saline (MOI = 103 vg/cell). Human AAT concentrations in culture media were assessed by ELISA. Each bar shows the average level of hAAT in treatment group (n = 3). (E–G) serum anti-hAAT antibody levels in NOD mice received a transplantation of dendritic cells ex vivo-infected with rAAV1-hAAT (E), rAAV8-hAAT (F), or saline (G). Each line shows anti-hAAT levels in an individual animal (n = 5).

Fig. 3.

Fig. 3.

Immune response to non-secretory transgene product in NOD mice. rAAV1-d-hAAT vector carries a hAAT gene with deletion of secretion signal sequence. (A) After infection of C2C12 cells with rAAV1-d-hAAT (d-hAAT, MOI = 103), hAAT protein can be detected by ELISA in the cells, but not in the culture medium. rAAV1- hAAT (hAAT) served as positive control group. Saline treated (PBS) cells as a negative control (n = 3). (B) hAAT levels in NOD mice injected with 2 × 1011 gv/mouse of rAAV1-CB-d-hAAT (rAAV1-d-AAT, n = 13), rAAV1-CB-hAAT (rAAV1-hAAT, n = 13), or injected with PBS (n = 13). Four weeks after vector injection, serum hAAT was detected by ELISA. (C and D) Anti-hAAT antibody levels were comparable in both rAAV1-CB-d-hAAT group (C) and rAAV1-CB-hAAT group (D). Each line shows the antibody levels in an individual animal (n = 13). (E and F) DC transplantation. Serum hAAT levels (E) and anti-hAAT antibody level (F) in NOD mice received DCs infected with rAAV1-d-hAAT or saline (MOI = 1013). Each line represents the average of hAAT or anti-hAAT antibody level in each group (n = 5).

Fig. 4.

Fig. 4.

Mouse cell produced transgene product does not generate an immune response. (A) Serum hAAT levels in NOD mice received a single injection of purified hAAT (Prolastin, 0.27 mg/mouse) or serum from hAAT-tg NOD mice (serum, 0.27 mg/mouse). Each line represents the average serum hAAT levels in each group detected by ELISA (n = 5). (B) Serum anti-hAAT levels. Note: no detectable anti-hAAT in mice receiving hAAT-tg NOD serum. Open square, Prolastin (purified hAAT); filed diamond, serum from hAAT-tg NOD mice.

Fig. 5.

Fig. 5.

Mouse cell produced transgene product induces transgene specific immune tolerance. (A and B) Cohorts of NOD mice (4 weeks of age) were IP infected with a high dose (2 × 1011 gv/mouse) of rAAV8-hAAT (open diamond, AAV8/AAV1, n = 3), low dose (2 × 1010 gv/mouse) of rAAV8-hAAT (open triangle, AAV8L/AAV1, n = 4), or IM injected with rAAV1-hAAT (2 × 1011 gv/mouse, filled circle, AAV1/AAV8, n = 7) or saline (filed triangle, PBS/AAV1, n = 5). Four weeks after the first injection, mice were infected with 2 × 1011 gv/mouse of another serotype of rAAV-hAAT. Serum hAAT levels (A) and anti-hAAT antibody levels (B) were detected by ELISA. Note: no antibody was detected in rAAV8-hAAT immunized groups. (C–E) In a separate experiment, two groups of NOD mice (n = 5) were IP injected with rAAV8-GFP vector (filed triangle, AAV8-GFP, 2 × 1010 vg/mouse) at 4 weeks of age or injected IP with 100 μL serum from hAAT–tg mice for 4 weeks (open diamond, serum contains 0.27 mg of hAAT, 2 injections/week). At 8 weeks of age, all mice were injected IP with rAAV1-hAAT (2 × 1011 vg/mouse). Serum hAAT levels (C) and anti-hAAT antibody levels (D) were detected by ELISA, and cellular immune response was evaluated by ELISpot (E). Note: mouse produced hAAT (Serum) injected group did not produce anti-hAAT antibody.

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