Sal-9RE7 Immunogenicity Evaluation

Sal-9RE7 was constructed by mixing 1 × 10⁸ CFU of heat-inactivated Salmonella to 1 ug 9RE7 and incubated with 1 × 10⁵ dendritic cells in 96 well plate cultured with complete RPMI media at 37°C, 5% CO2 overnight. After aspirating culture medium and washing with PBS, 5 × 10⁵ E7-specific CD8+ T cells were added to dendritic cell line and blocked with Brefeldin A + Monensin Golgi Plug (Thermo Fisher Scientific) overnight.^{22 (link),23 (link)} Cells were collected and stained with PE-conjugated HPV16 E7aa49–57 peptide loaded H-2 Db E7 tetramer and APC-A750-conjugated anti-mouse CD8α antibody (Biolegend) before permeabilization with eBioscience Fixation (Invitrogen). FITC-conjugated IFNγ antibody was used for intracellular staining.^{24 (link)}