F;actors influencing cross-presentation of non-self antigens expressed from recombinant adeno-associated virus vectors

Background We have previously demonstrated that recombinant adenoassociated virus vectors expressing the influenza virus hemagglutinin (rAAV-HA) in sk eletal muscle results in T-cell priming and muscle fiber destruction due to cross-presentation of HA by dendritic cells (DC). Based on controversial o bservations concerning the stability of non-self proteins expressed from rA AV vectors it is important to understand the factors influencing cross-pres entation of transgene products following rAAV mediated gene transfer, in or der to be able to use this vector safely in the clinic. Methods In order to understand the factors influencing in vivo cross-presen tation of non-self proteins, we have retargeted the immunogenic lacZ protei n in the context of rAAV from the cytoplasm to the cell surface and studied the activation of LacZ specific immune responses following intramuscular m ediated gene transfer. In addition, using tools available for studying in v itro 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 t he nature of the cross-presented material. Results 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 cr oss-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 resulte d in target cell destruction, which is in stark contrast to the stability o bserved with rAAV-LacZ vectors expressing the cytoplasmic form of LacZ. Usi ng an in vitro assay, we were able to show that dendritic cells (DC) in add ition to B-cells cross-presented HA to class II restricted T-ceIls whereas only the former were able to activate class I restricted CD8 + T-cells. Hig h-dose antigens were needed for efficient class I restricted T-cell priming , whereas class II restricted T-fells were activated by less antigen. Conclusion The present results indicate that immune responses to non-self a ntigens expressed from rAAV Vectors depend on the accessibility of such ant igens to different local antigen-presenting cells. Copyright (C) 2001 John Wiley & Sons, Ltd.