Role of Vector in Activation of T Cell Subsets in Immune Responses against the Secreted Transgene Product Factor IX

Abstract

Defining immune responses against the secreted transgene product in a gene therapy setting is critical for treatment of genetic diseases such as hemophilia B (coagulation factor IX deficiency). We have previously shown that intramuscular administration of an adeno-associated viral (AAV) vector results in stable expression of therapeutic levels of factor IX (F.IX) and may be associated with humoral immune responses against F.IX. This study demonstrates that intramuscular injection of an AAV vector expressing F.IX fails to activate F.IX-specific cytotoxic T lymphocytes (CTLs) in hemostatically normal or in hemophilia B mice, so that there is an absence of cellular immune responses against F.IX. However, transgene-derived F.IX can cause B cell responses characterized by production of T helper cell-dependent antibodies (predominantly IgG1, but also IgG2 subclasses) resulting from activation of CD4+ T helper cells primarily of the Th2 subset. In contrast, administration of an adenoviral vector efficiently activated F.IX-specific CTLs and T helper cells of both Th1 and Th2 subsets, leading to inflammation and destruction of transduced muscle tissue and activation of B cells as well. Therefore, vector sequences fundamentally influence T cell responses against transgene-encoded F.IX. In conclusion, activation of the immune system in AAV-mediated gene transfer is restricted to pathways mediated by F.IX antigen presentation through MHC class II determinants resulting in T and B cell responses that are more comparable to responses in the setting of protein infusion rather than of viral infection/gene transfer.

Major role of local immune responses in antibody formation to factor IX in AAV gene transfer

“…Similarly, our new data with AAV-2 vector (which in our hands is approximately 10-to 20-fold less efficacious than serotype 1 vector in muscle) show emerging hF.IX antigen levels by 7-8 weeks at a dose of 4 Â 10 13 vg/kg, which is not seen at lower AAV-2 vector doses. 8,9,14 Taken together, these results indicate that F.IX antigen levels produced by transduced muscle tissue are a critical parameter that determines whether neutralization of systemic expression by antibody formation is sustained. Levels of expression o0.2 mg/ml are typically not sufficient to overcome the antibody response, while higher levels (0.2-3.5 mg/ml) may promote decline of the antibody response over time.…”

“…However, this did not result in an inflammatory response, likely because of inefficient CD8 + T-cell activation. 9,14,23,24 It is thought that because of unproductive transduction of dendritic cells and lack of innate immunity to the AAV vector, there is inefficient MHC class I presentation of F.IX peptides to CD8 + T cells by professional antigen presenting cells (APCs). [23][24][25] However, lack of inflammation during the first month after gene transfer and shift toward Th2 immunity did not prevent subsequent activation of CD8 + T cells and inflammation at later time points (Figure 3).…”

“…In contrast to adenoviral gene transfer, the ensuing inflammation caused only partial loss of transgene expressing cells, which may be explained by lower efficiency in activation of CTLs, less efficient Th1 help, or increase of transgene expression over time (secondary to slow uncoating of AAV-2 vector particles). 9,23,34 Interestingly, the animal with the sharpest decline in transgene expression had a late increase in IgG2a production, a Th1-dependent subclass of immunoglobulin, suggesting a late increase in Th1 immunity, which can be correlated with the inflammatory response. Given that the inflammatory reaction occurred long after gene transfer, and that these animals are not subject to natural infection with AAV, CD8 + T-cell responses were most likely directed against the hF.IX antigen.…”

“…6 However, several studies demonstrated formation of antibodies to the F.IX transgene product after AAV-mediated gene transfer. [7][8][9][10][11][12] The risk for such immune responses depended on the underlying F.IX mutation, route of vector administration, vector dose and serotype. 13 Inhibitor formation to F.IX was most frequently observed after intramuscular (i.m.)…”

“…15 Anti-F.IX formation upon muscle gene delivery is due to an adaptive, CD4 + T-celldependent immune response which is absent in CD4-or Rag1-deficient mice. 9,14 Transient immune suppression using cyclophosphamide successfully prevented inhibitor formation. 8,10,11 In contrast, sustained expression of canine F.IX in hemophilia B dogs with a F.IX missense mutation was obtained after i.m.…”