Mice work
Male BALB/c mice aged 6-8 weeks were purchased from SLAC, Shanghai and housed in Experimental Animal Centre, Fudan University. Male C57BL/6 mice aged 6-8 weeks transgenic for Cre recombinase (Cre Tg C57BL/6) has been described previously [14]. To construct BPS persistence mice, BALB/c mice were injected with 10 μg of BPS plasmids through HDI method via tail vein and selecting mice positive for serum HBV antigens for 4 weeks. To construct rcccDNA persistence mice, Cre Tg C57BL/6 mice were injected with 1.5 × 109 plaque-forming units (PFU) of Adv/prcccDNA in 200 μl PBS via tail vein and selecting mice positive for serum HBV antigens for 4 weeks. For evaluating the therapeutic effects of IL-21, HBV persistence mice were injected with 10 μg of LNP-IL-21 or control LNP in 200 μl PBS via tail vein, and again two weeks later. Blood samples, liver and spleen tissues were collected at indicated time points for further analysis.
For ex vivo LNP-IL-21 stimulation, PBMC isolated from BPS persistence mice were cultured in RPMI containing 2 mM L-glutamine, 50 U/ml penicillin, and 10% fetal bovine serum (all from Invitrogen, China) and transfected with 1 μg of LNP-IL-21 or LNP. At 6 hours post transfection, recombinant HBsAg (rHBsAg) was added in culture media (5 μg/mL). rHBsAg was kindly provided by Prof. Di Qu, Fudan University. At 3 days post rHBsAg stimulation, 1 × 106 cells were injected into each treatment-naïve BPS persistence mice via tail vein. Mouse handling procedures were approved by the Animal Ethics Committee of School of Basic Medical Sciences, Fudan University. The approval number was JS-028.