LNP-IL-21 treatment successfully indued potent anti-HBV immune responses
LNP-IL-21 induced HBV clearance in both BPS and rcccDNA persistence mouse models was associated with HBsAb seroconversion (Figure 2B and 4B ), suggesting LNP-IL-21 treatment is probably able to activate HBsAg-specific humoral immune responses. To prove it, at 8 w.p.i., PBMC were collected from LNP-IL-21-cured BPS mice achieving HBsAb seroconversion (n = 6) and LNP-injected mice (5 out 6 mice positive for HBsAg), and subjected to flow cytometry analysis for HBsAg-specific B cells (Figure 6 A, C-D ). The percentages of HBsAg-specific B cells in LNP-IL-21-cured mice were significantly higher than those in LNP-injected mice (Figure 6 E ), and representative images of two mice in each group were presented inFigure 6F .
On the other hand, ALT transient elevations were observed in above two mouse models post LNP-IL-21 injection (Figure 2E and 4E ), indicating acute liver injury. In order to investigate whether LNP-IL-21 treatment participated in activation of cellular immune responses, which is responsible for the removal of HBV-harboring hepatocytes to achieve cure, mouse liver tissues were collected form LNP-IL-21-and LNP-injected mice at 2 and 8 w.p.i., and then subjected to H&E and anti-CD8 staining (Figure 6 A, ). As shown in Figure 6B , marked intrahepatic immune cell infiltration, including CD8+cell infiltration, was observed in LNP-IL-21-injected mice at 2 w.p.i., but not at 8 w.p.i., which roughly coincided with HBV clearance. No obvious immune infiltration was observed in LNP-injected mice at 2 or 8 w.p.i.. What’s more, at 8 w.p.i., splenocytes from LNP-IL-21-cured mice, but not LNP-injected mice, secreted much more IFN-γ post HBcAg or HBsAg stimulation (Figure 6G ), indicating activated T cell immune responses. In a summary, LNP-IL-21-based gene therapy successfully induced both potent anti-viral humoral and cellular immune responses contributing to HBV clearance.