<jats:title>Abstract</jats:title> <jats:p>Previously a well-defined dendritic cell (DC)-targeting strategy of anti-DEC205 based vaccine shows highly enhanced CD4+ T cell immunity. We have demonstrated that a DNA vaccine with soluble (s)PD1 fused with HIV-1 p24 antigen can greatly enhance p24-specific humoral and cellular immune responses in mice by targeting DCs. Consequently, we compared the efficacies of the two vaccines in our system and found that sPD1-based DNA vaccination induced higher functional CD8+ T cell responses. Although the binding and uptake of encoded fusion proteins are similar between sPD1-p24 and anti-DEC205-p24 in DCs in vitro, transfer of DCs pulsed with these proteins into mice resulted in the sPD1-p24 group eliciting higher IFN-γ releasing CD8+ T cells. These data suggests that different mechanistic pathways exist between the two DC-targeting strategies. We examined the route of antigen processing/presentation by confocal microscopy and found that sPD1-p24 protein co-localized to both Rab14 and Lamp1 endosomal compartments used for MHC class I and II presentation, respectively, while anti-DEC205-p24 was only found in the latter. In addition, sPD1 DNA vaccination resulted in draining lymph node DCs with elevated CD40 and MHC class II expression with higher production of IL-12 compared to anti-DEC205. The mechanism of sPD1-based vaccination in enhancing CD8+ T cells immunity is likely dependent on DC-targeting and activation, production of Th1 cytokines and antigen cross-presentation.</jats:p>