Cytosolic delivery of HBsAg and enhanced cellular immunity by pH-responsive liposome

It remains a major challenge to prime the cytotoxic T lymphocytes (CTLs) response for the treatment of Hepatitis B virus (HBV) infection. Inspired by an important natural biological behavior, membrane fusion, we constructed a pH-responsive nanocarrier (HBsAg&CpG@Lip) with a membrane fusion capacity for HBsAg intracellular delivery and subsequent process of CTLs. In the in vitro experiments, HBsAg&CpG@Lip greatly promoted the cellular uptake of HBsAg and the activation of BMDCs. It induced the cytosolic release of HBsAg in BMDCs, which was in conformity with the membrane fusion capacity of HBsAg&CpG@Lip. Such a capacity was improved by 4.4-fold in pH = 5.0 than that in pH = 7.4 at 60 min, which was calculated through fluorescence resonance energy transfer (FRET) efficiency. Furthermore, the cross-presentation activity induced by liposome at 1 μg/mL was equivalent to that by soluble antigen at 2000 μg/mL, which indicated an advantage for the liposome in cytosolic antigen delivery and potent CTLs activation. In respect of the transport of liposomes into draining lymph nodes (DLNs), the recruitment of liposome⁺migratory DCs at 24 h raised 11-fold than that at 3 h, suggesting that liposomes were mainly carried by migratory DCs. Meanwhile, HBsAg&CpG@Lip also elicited the activation of DCs and the generation of T_fh cells in DLNs. After immunization, HBsAg&CpG@Lip induced a higher anti-HBsAg IgG and ratio of IgG2c/IgG1 than that by HBsAg or Alum+HBsAg group. It also augmented a strong CTLs response as expected. Specifically, stimulation with HBsAg&CpG@Lip increased the number of IFN-γ-secreting splenocytes, the proportion of CD107α⁺CD8⁺and FasL⁺CD8⁺ T cells and the secretion of Granzyme B, which verified the design of membrane fusion in vivo. In summary, this design for HBsAg cytosolic delivery exhibits great benefits for triggering CTLs, opening an avenue for the development of a potent therapeutic HBV vaccine.