Factors influencing cross-presentation of non-self antigens expressed from recombinant adeno-associated virus vectors

We have previously demonstrated that recombinant adeno-associated virus vectors expressing the influenza virus hemagglutinin (rAAV-HA) in skeletal muscle results in T-cell priming and muscle fiber destruction due to cross-presentation of HA by dendritic cells (DC). Based on controversial observations concerning the stability of non-self proteins expressed from rAAV vectors it is important to understand the factors influencing cross-presentation of transgene products following rAAV mediated gene transfer, in order to be able to use this vector safely in the clinic.

In order to understand the factors influencing in vivo cross-presentation of non-self proteins, we have retargeted the immunogenic lacZ protein in the context of rAAV from the cytoplasm to the cell surface and studied the activation of LacZ specific immune responses following intramuscular mediated gene transfer. In addition, using tools available for studying in vitro HA-specific T-cell activation, our aim was to identify the cell types involved in class I and class II restricted cross-presentation as well as the nature of the cross-presented material.

By retargeting the lacZ protein in the context of rAAV to the cell membrane, we found that one of the factors influencing the efficiency of cross-presentation of non-self antigens is the localization of the transgene product within the target cells. Following rAAV-LacZ mediated gene transfer to the muscle we demonstrated that the membrane-bound form of LacZ resulted in target cell destruction, which is in stark contrast to the stability observed with rAAV-LacZ vectors expressing the cytoplasmic form of LacZ. Using an in vitro assay, we were able to show that dendritic cells (DC) in addition to B-cells cross-presented HA to class II restricted T-cells whereas only the former were able to activate class I restricted CD8+ T-cells. High-dose antigens were needed for efficient class I restricted T-cell priming, whereas class II restricted T-cells were activated by less antigen.

The present results indicate that immune responses to non-self antigens expressed from rAAV vectors depend on the accessibility of such antigens to different local antigen-presenting cells.