Efficient generation of cytotoxic T lymphocytes against cervical cancer cells by adeno-associated virus/human papillomavirus type 16 E7 antigen gene transduction into dendritic cells

Abstract

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.

T-cell responses to human papillomavirus type 16 among women with different grades of cervical neoplasia

“…Studies using whole HPV proteins to restimulate CTL responses, either in a soluble form (Nakagawa et al, 1997) or expressed by recombinant viral vectors (Nimako et al, 1997), where the whole protein is naturally processed, have been more successful. Autologous dendritic cells (DCs) presenting HPV16 peptides or whole proteins have also been used to restimulate E6-and E7-specific CTL responses from both normal donors and women with cervical carcinoma in vitro (Murakami et al, 1999;Schoell et al, 1999;Thornberg et al, 2000;Davidson et al, 2001;Liu et al, 2001;Chiriva-Internati et al, 2002;Valdespino et al, 2005), although it is not clear whether these represent primary or memory T-cell responses. Overall, the results suggest that naturally occurring HPV E6-and E7-specific CTLs do exist in patients with HPV-associated disease, although their detection may depend on the methods used to reactivate them in vitro.…”

Adeno-associated virus vectors: potential applications for cancer gene therapy

“…In addition, high levels of CD8 þ T cells were induced by transduced DC with high levels of CD80. 148,149 Based these observations, AAV-transduced tumor cells or DCs induce very strong CTL response to tumor cells.…”

Immune responses to adeno-associated virus and its recombinant vectors

“…rAAV vector-based genetic vaccines can successfully induce primary humoral and cellular immune responses directed against specific antigens in animal models [7][8][9] and in vitro cultures with human lymphocytes. [10][11][12] Thus, as in most biological systems, there is a delicate balance between the generation of desirable and unwanted effects.…”