AAV vectors have long been regarded as having little potential to induce a CTL response because they do not contain any viral genes. However, it has been suggested that injection of AAV2 vectors may actually result in the generation of de novo
CTL responses elicited by the input particles or in the activation of a memory T cell response due to prior AAV exposure (Manno et al.
). In a clinical trial, patients treated with an AAV-factor IX vector were found to express factor IX only transiently and the decline in expression correlated with a rise in serum transaminase levels and the appearance of capsid-specific T cells. On the basis of these observations, it was hypothesized that the loss of expression was due to the induction of an AAV-specific CTL response leading to the destruction of transduced liver cells. In view of these observations, it becomes important to better understand the role that the host immune response plays in affecting transgene expression after the administration of AAV vectors. Although much attention has been focused on the possible involvement of AAV-specific CTLs, it is important to consider that host immune responses can be directed against not only the viral vector itself but also the transgene being expressed and can consist of both T and B cell components.
In this study, we have explored the relative contribution of B and T cell responses as well as the impact of responses directed against the AAV capsid and the transgene product on AAV-mediated expression. Our results confirm that neutralizing antibodies against AAV2 can prevent transduction and subsequent transgene expression by an AAV2/α-Gal vector. Analysis of CTL responses indicated that a CTL response against the α-Gal transgene product is capable of limiting transgene expression whereas CTLs against AAV capsid have little impact on the magnitude or duration of expression.
Adenoviral and plasmid expression systems were used to create immune responses against the Cap protein of AAV2. In immune-competent C57BL/6 mice primed with plasmid encoding Cap, both antibody and CTL responses developed against the AAV2 capsid protein and no transgene expression was detected, making it difficult to determine whether T cells, B cells, or a combination of the two was responsible for affecting AAV transduction and subsequent transgene expression. The presence of neutralizing antibodies to AAV has been previously reported to negatively influence AAV transduction, resulting in low levels of transgene expression (Scallan et al., 2008). To assess the role of Cap-specific CTLs in the absence of neutralizing antibodies, the experiment was repeated in B cell-deficient μMT mice, which developed a strong CTL response to the AAV capsid protein but no antibodies. Under these conditions, the Cap-specific CTL response in μMT mice failed to affect the strength or duration of transgene expression. This would suggest that in immunocompetent mice, Cap-specific CTLs did not affect transgene expression but that neutralizing antibodies effectively prevented transduction with AAV vector. These results are consistent with the reports of C. Li and colleagues and Wang and colleagues, who demonstrated that factor IX expression remains constant in the presence of capsid-specific CTLs. Therefore, although CTLs can be raised against the AAV Cap protein and may be detected in the host, they are unlikely to be the main contributing factor in the loss of transgene expression.
Comparatively little attention has been paid to the potential role of CTLs against the transgene product in limiting expression from AAV vectors. The use of AAV for gene therapy delivery of a protein that is either truncated or not expressed in a patient may result in the generation of a therapy-specific immune response. In these cases, T cell responses induced by the transgene may significantly impact the overall therapeutic benefit of the gene therapy being used. For proof-of-concept experiments, we have chosen to use the α-Gal transgene as a way to mimic the delivery of a therapeutic protein against which the patient is not immunologically tolerized. Our results demonstrate that transgene-specific CTLs have a direct effect on the level and duration of expression from an AAV vector. This was clearly demonstrated by experiments in which B cell-deficient μMT mice were preimmunized with Ad2/α-Gal vector to generate CTLs, but no antibodies, against α-Gal. When subsequently injected with AAV2/α-Gal vector, these mice exhibited a more than 2-log decrease in initial levels of transgene expression compared with unprimed mice or mice that were preimmunized with Ad5/Cap to generate CTLs to the capsid protein. Within 2 weeks, circulating levels of α-Gal protein were undetectable, indicating that transgene expression was severely compromised by the presence of a CTL response against the transgene product whereas Cap-specific CTLs had no impact. Similar to results observed in the hemophilia clinical trial, the decrease in circulating levels of therapeutic protein was accompanied by an elevation in serum transaminase levels.
It remains unclear why preformed CTLs against the transgene product are capable of terminating transgene expression whereas CTLs against Cap, when present at equivalent levels of lytic activity (as measured in vitro), have no measurable impact on transgene expression. One possible explanation may reside in the degree of antigen presentation required for sensitization of target cells to the lytic activity of specific CTLs. After AAV vector administration, there is abundant transgene expression that should result in robust presentation on the cell surface in association with MHC class I. However, the Cap protein is not expressed by the vector and transduced hepatocytes must process and present capsid protein from the initial input number of viral particles by a nonclassical method of MHC class I presentation. It is conceivable that the amount of processed capsid peptide presented on the cell surface is not sufficient to render the cells susceptible to lysis by capsid-specific CTLs. It has also been hypothesized that murine hepatocytes may process and present antigen less efficiently than human hepatocytes, explaining observed differences between mouse experiments and the hemophilia clinical trial described previously. On the basis of our results, α-Gal-specific CTLs appear capable of directly affecting transduced cells and abolish transgene expression, suggesting that the processing and presentation pathway is fully functional in transduced murine hepatocytes.
Results from our studies have potential implications for the clinical application of AAV-based gene therapy. Although preexisting CTLs against AAV appear unlikely to represent a significant impediment to transgene expression, the induction of CTLs against the transgene product potentially represents a hurdle in the clinic. For many genetic diseases such as hemophilia, the goal of gene therapy is to introduce a functional protein into patients who either do not express the protein, express a truncated version, or express nontherapeutic levels of the protein. During the course of T cell development, these patients may not become tolerized to the wild-type protein because of this alteration in or lack of expression. Therefore, the likelihood exists that T cell responses to these proteins may affect long-term expression from a gene therapy vector. In such instances, the therapeutic protein expressed by the vector may be recognized as a nonself antigen, resulting in the induction of a destructive CTL response. To circumvent the potential effect of CTL responses against the transgene product, it may be necessary to develop strategies to tolerize patients against the therapeutic protein before AAV vector administration or to establish transient immunosuppression regimens that can be delivered at the time of vector administration to minimize the generation of CTL responses.