Off-the-shelf adenoviral-mediated immunotherapy via bicistronic expression of tumor antigen and iMyD88/CD40 adjuvant.

Recent modest successes in ex vivo dendritic cell (DC) immunotherapy have motivated continued innovation in the area of DC manipulation and activation. Although ex vivo vaccine approaches continue to be proving grounds for new DC manipulation techniques, the intrinsic limits of ex vivo therapy, including high cost, minimal standardization, cumbersome delivery, and poor accessibility, incentivizes the development of vaccines compatible with in vivo DC targeting. We describe here a method to co-deliver both tumor-specific antigen (TSA) and an iMyD88/CD40 adjuvant (iMC), to DCs that combines toll-like receptor (TLR) and CD40 signaling. In this study, we demonstrate that simple TSA delivery via adenoviral vectors results in strong antitumor immunity. Addition of iMC delivered in a separate vector is insufficient to enhance this effect. However, when delivered simultaneously with TSA in a single bicistronic vector (BV), iMC is able to significantly enhance antigen-specific cytotoxic T-cell (CTL) responses and inhibit established tumor growth. This study demonstrates the spatial-temporal importance of concurrent DC activation and TSA presentation. Further, it demonstrates the feasibility of in vivo molecular enhancement of DCs necessary for effective antitumor immune responses.

Tobacco mosaic virus efficiently targets DC uptake, activation and antigen-specific T cell responses in vivo.

Over the past 20 years, dendritic cells (DCs) have been utilized to activate immune responses capable of eliminating cancer cells. Currently, ex vivo DC priming has been the mainstay of DC cancer immunotherapies. However, cell-based treatment modalities are inherently flawed due to a lack of standardization, specialized facilities and personnel, and cost. Therefore, direct modes of DC manipulation, circumventing the need for ex vivo culture, must be investigated. To facilitate the development of next-generation, in vivo targeted DC vaccines, we characterized the DC interaction and activation potential of the Tobacco Mosaic virus (TMV), a plant virus that enjoys a relative ease of production and the ability to deliver protein payloads via surface conjugation. In this study we show that TMV is readily taken up by mouse bone marrow-derived DCs, in vitro. Footpad injection of fluorophore-labeled TMV reveals preferential uptake by draining lymph node resident DCs in vivo. Uptake leads to activation, as measured by the upregulation of key DC surface markers. When peptide antigen-conjugated TMV is injected into the footpad of mice, DC-mediated uptake and activation leads to robust antigen-specific CD8(+) T cell responses, as measured by antigen-specific tetramer analysis. Remarkably, TMV priming induced a greater magnitude T cell response than Adenovirus (Ad) priming. Finally, TMV is capable of boosting either Ad-induced or TMV-induced antigen-specific T cell responses, demonstrating that TMV, uniquely, does not induce neutralizing self-immunity. Overall, this study elucidates the in vivo DC delivery and activation properties of TMV and indicates its potential as a vaccine vector in stand alone or prime-boost strategies.