For LN delivery studies, C57BL/6 mice were injected with soluble Cy5-CPG + CSIINFEK(FITC)L and Cy5-CPG NP/CSIINFEK(FITC)L. After 12 hours, inguinal LNs were harvested, and a regular LNs fluorescence imaging was conducted using Maestro optical imaging system. Subsequently, LNs were dissociated in collagenase D and DNase I according to the manufacturer’s instructions. Single cells were collected after dissociation and then stained with CD11c for DCs and F4/80 for macrophages. In vivo delivery of banNVs into APCs was analyzed by a BD Beckman Coulter flow cytometer. For assessment of the elicited antigen-specific T cell responses, C57BL/6 mice were injected with different formulations of vaccines on days 0, 7, and 14. Peripheral blood cells were collected on day 21. First, red blood cells were lysed using ACK lysis buffer for 5 min and then blood clot was removed by gentle centrifugation. Next, the left cells were stained with 4′,6-diamidino-2-phenylindole in fetal calf serum buffer for 10 min to block the Fc segment. Then, cells were stained with mixed antibodies including anti–CD8-APC-Cy7, dextramer-PE (Immudex), and PD1-BV421 for 1 hour on ice. Cells were subsequently washed three times and resuspended in Cytofix for 20 min at 4°C. Last, cells were washed with Perm/Wash buffer twice for flow cytometry quantification using a BD LSRFortessa X-50 flow cytometer.

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