612. Determination of Specific Th1 and Th2 CD4 T Cell Responses in AAV2- and AAV8-Mediated Human Factor IX Gene Therapy in Mice

Abstract

Recombinant AAV is promising for coagulant factor IX (F.IX) gene transfer due to the long-term expression of hF.IX in the liver and weak immune response using a liver specific promoter. However, in humans and some animals the F.IX expression declines over long time periods and immune responses to vector and transgene can occur. It was shown that C3H (H2k) mice express the highest levels of anti-hF.IX antibodies, which requires the IAka and IAk b and the IL-10 region of Chromosome 1 (Blood 105:1029). We utilized MHC class II peptides in the context of I-Ak and I-Ek to specifically detect Th cells in AAV-hF.IX immunized C3H mice. First, we use a prediction program, SYPFEITHI, to predict MHC II binding peptides from hF.IX, AAV2 and AAV8 capsid proteins (Table 1). C3H mice were iv injected with 2|[times]|1011vg of AAV2- or AAV8-hF.IX, in which hF.IX expression is regulated by a liver-specific ApoE/hAAT promoter. The mice were boosted 30 days later. Blood samples were collected for analysis of ALT and plasma hF.IX. The plasma ALT level was significantly elevated (p<0.05) at day 9 after boost with AAV2- or AAV8-hF.IX. There was no significant decrease in plasma hF.IX levels up to 9 days after boost. To determine the frequency of hF.IX, AAV2 and AAV8 specific CD4 Th cells in liver, mononuclear cells (MNC) were prepared and analyzed by peptide specific ELISPOT assays for production of Th1 cytokine IFN|[gamma]| and Th2 cytokine IL-4, respectively. Liver MNCs were co-cultured with irradiated antigen presenting cells pulsed with the peptides specific for hF.IX, AAV2 or AAV8. The number of IFN|[gamma]| spot forming cells (SFC) was determined 48 hr later (Table 1). For both AAV2-hF.IX and AAV8-hF.IX infected mice, an I-Ak restricted hF.IX epitope AA108-122 induced both IL-4 and IFN|[gamma]| production. In AAV2-hF.IX infected mice, an I-Ek restricted AAV2 epitope AA475-489 induced more IL-4 SFCs than IFN|[gamma]| SFCs (p<0.05). For AAV8-hF.IX infected mice, an I-Ek restricted AAV8 epitope AA126-140 was found to induce more IFN|[gamma]| SFCs than IL-4 SFCs (p<0.05). Similar results were seen in spleen of immunized mice. Other predicted peptides failed to induce IFN|[gamma]| or IL-4 SFCs in immunized mice (data not shown). These results indicated that AAV2- or AAV8-hF.IX gene transfer induced both vector and transgene specific Th cells with the highest response in IFN|[gamma]| production to hF.IX epitope peptide AA108-122. Moreover, these epitope peptides could function differently on Th1 and Th2 cells to drive a Th response towards CD8 T cell response or B cell activation. These findings will facilitate a better understanding in CD8 T cell response or antibody response to AAV-hF.IX gene therapy.