LNP-IL-21 induced HBV clearance in BPS-based persistence mouse model
To investigate whether LNP-IL-21 had any therapeutic effects against HBV persistence, BALB/c mice displaying persistently positive serum HBV antigens up to 4 weeks post injection of BPS were subjected to two-dose LNP-IL-21 injections, two weeks apart, or control LNP via tail vein. Serum HBV markers and biochemical indexes were regularly measured every 2 weeks up to 8 weeks post injection of LNP (w.p.i.). As shown inFigure 2 A and C , LNP-IL-21 administration caused HBsAg and HBeAg levels to decrease much more quickly compared to mice (n = 6) receiving LNP injection, and become undetectable in all LNP-IL-21-injected mice (n = 6) by 6 w.p.i., while both viral markers persisted in 5 out of 6 LNP-injected mice. Meanwhile, all LNP-IL-21-injected mice which had cleared serum HBsAg displayed HBsAb seroconversion by 6 w.p.i. (Figure 2B ), indicating anti-HBsAg humoral responses was involved in HBsAg clearance. Accordingly, HBV DNA in pooled serum started to decrease following LNP-IL-21 injection and dropped to detection limit level by 8 w.p.i., while it remained largely unchanged in LNP-injected group (Figure 2D ). LNP-IL-21-injected mice manifested ALT and IL-21 elevations at 3 w.p.i., which reverted to normal levels by 6 and 8 w.p.i. respectively (Figure 2E-F ). In contrast, no obvious changes in ALT or IL-levels were detected in LNP-injected mice (Figure 2E-F ). Total bilirubin and creatine kinase levels were generally normal in both two groups (Figure S4 ).
In addition to serum HBV markers analyses, both intrahepatic HBV DNA and capsids were assayed using Southern and Western blots to confirm true clearance at 8 w.p.i. (Figure 3A) . As shown in Figure 3B , HBV replication intermediates, BPS replicons DNA or capsids were undetectable in all LNP-IL-21-injected mice (n = 6) that had cleared serum HBsAg, but persisted in 5 out of 6 LNP-injected mice. Collectively, LNP-IL-21 efficiently and safely induced HBV clearance in BPS-based persistence mouse model.