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We have shown that the pulsing of dendritic cells ( DCs ) with human papillomavirus type 16 ( HPV -16 ) antigen proteins by lipofection stimulates class I -restricted cytotoxic T lymphocyte ( CTL ) response against primary cervical cancer cells. Also, we have shown that adeno -associated virus ( AAV ) was able to effectively deliver a cytokine gene into DCs. It has been our hypothesis that the delivery of antigen genes into DCs, resulting in endogenous and continuous antigen protein expression, may result in an improvement in T -cell priming by DCs. Here, DCs are pulsed ( infected ) with an AAV vector containing the HPV -16 E6 gene. After infection, transduced E6 gene mRNA expression and vector chromosomal integration could be identified in infected DCs. Furthermore, priming rosettes formed at early times when the AAV / E6 vector was used. Most importantly, AAV / E6 vector pulsing of DCs induced, after only 7 days of priming, a strong CTL response against primary cervical cancer cell lines, compared to bacterial E6 protein lipofection. Killing was significantly blocked by the addition of anti -MHC class I antibodies. Fluorescence -activated cell sorter ( FACS ) analysis of resulting primed cell populations revealed higher levels of CD8 + T cells by AAV -based pulsing, with little evidence of CD56 ( NK ). FACS analysis of the DC populations revealed that AAV / E6 vector -pulsed DCs had higher levels of CD80 and lower levels of CD86 than protein -pulsed DCs. These data suggest that rAAV may be appropriate for antigen pulsing of DCs for immunotherapy protocols. Finally, our protocol represents an advance in regards to the time needed for generating a CTL response compared to other techniques. Cancer Gene Therapy ( 2001 ) 8, 948 -957

Adeno‐associated virus (AAV) is able to efficiently deliver a cytokine gene into dendritic cells (DC). Improvements in T cell priming by DC might be effected by the delivery of antigen genes into DC, resulting in continuous protein expression, as most proteins have short half‐lives. In this study, a recombinant AAV vector containing the human papillomavirus (HPV)‐16 E7 gene was used to pulse/infect DC and compared to the pulsing of DC by the lipofection of bacterially produced E7 protein. Pulsing of DC with AAV/antigen (Ag) gene was found to be superior to pulsing with protein in six different assay systems: (1) the level of antigen transfer into DC as determined by intracellular staining; (2) the level of MHC class I‐restricted killing in cytotoxic T lymphocyte (CTL) assays;(3) the level of IFN‐γ expression; (4) the level of DC‐T cell priming clusters generated; (5) the level of CD80 and CD83 expression on DC; and (6) in the resulting CD8:CD4 ratio. Finally, AAV/Ag gene pulsing resulted in strong CTL activity after only 7 days of priming. These data suggest that AAV vectors may offer advantages over the commonly used protein‐pulsing technique and that AAV vectors may be useful for the stimulation of CTL activity and adoptive immunotherapy protocols.

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