Anti–PD-L1–IFN-α–adjuvanted HBsAg vaccine overcomes HBV immune tolerance through targeting both DCs and macrophages
Chao-Yang Meng, Yong Liang, Longxin Xu, Hongjia Li, Jingya Guo, Hairong Xu, Fan Wang, Yang-Xin Fu, Hua Peng
Chao-Yang Meng, Yong Liang, Longxin Xu, Hongjia Li, Jingya Guo, Hairong Xu, Fan Wang, Yang-Xin Fu, Hua Peng
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Research Article Hepatology Immunology Virology

Anti–PD-L1–IFN-α–adjuvanted HBsAg vaccine overcomes HBV immune tolerance through targeting both DCs and macrophages

Abstract

Recombinant hepatitis B surface antigen (rHBsAg) vaccine with various adjuvants fails to break T and B cell tolerance in hosts with chronic hepatitis B (CHB). This study aims to explore the mechanisms to break immune tolerance that allows the host to respond to rHBsAg, achieving a cure for CHB. We engineered an anti–PD-L1–IFN-α (aPD-L1–IFN-α) heterodimeric fusion protein to allow rHBsAg to rejuvenate T and B cell responses in hepatitis B virus–tolerant (HBV-tolerant) mice. S.c. coimmunization with aPD-L1–IFN-α and rHBsAg significantly enhanced antigen uptake and maturation of both macrophage and dendritic cell (DC) subsets in draining lymph nodes. Macrophages drove early B cell activation, while cDC1s primed CD8+ T cells, breaking tolerance and leading to both B cell and cytotoxic T lymphocyte (CTL) differentiation. This strategy elicited not only anti-HBsAg neutralizing antibodies but also HBsAg-specific CD8+ T cell responses, achieving a functional cure without systemic toxicity. The efficacy of the aPD-L1–IFN-α adjuvant depended on both PD-L1 cis-targeting and IFN-α receptor signaling in antigen-presenting cells. These findings establish aPD-L1–IFN-α as a translatable adjuvant to break the strong tolerance induced by CHB, providing a dual-pathway strategy to induce HBV-specific T and B cell responses.

Authors

Chao-Yang Meng, Yong Liang, Longxin Xu, Hongjia Li, Jingya Guo, Hairong Xu, Fan Wang, Yang-Xin Fu, Hua Peng

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Figure 4

aPD-L1–IFN-α breaks immune tolerance to rHBsAg vaccine through coordinating DCs and macrophages.

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aPD-L1–IFN-α breaks immune tolerance to rHBsAg vaccine through coordinat...
(A–C) HBV carrier CD11c-DTR bone marrow chimeric mice were generated and treated as described in Figure 3A. For DC depletion, DT (4 ng/g body weight) was administered i.p. every 2 days starting 24 hours before immunization. Serum levels of ayw subtype–specific anti-HBsAg IgG (A) and HBsAg (B) (n = 4–5/group) were determined by ELISA. The detection limit in B is indicated by a dashed line. A total of 1 × 106 splenocytes from each mouse (n = 4–5/group) were collected on day 126. Specific B cell responses to HBsAg (subtype ayw) were tested by a B cell ELISpot assay (C). (D and E) CD11c-DTR bone marrow chimeric mice (n = 5/group) were immunized s.c. with the mixture of aPD-L1–IFN-α (2 μg/mouse) and rHBsAg (1 μg/mouse) on days 0 and 14. For DC depletion, DT (4 ng/g body weight) was administered i.p. every 2 days starting 24 hours before immunization. HBsAg-specific IgG (D) and IgM (E) responses were measured on day 21 and day 10 after the first immunization, respectively. (F–H) HBV carrier mice were treated as described in Figure 3A. For macrophage depletion in inguinal LNs, CLL (40 μL/mouse) was injected s.c. every 3 days, starting 7–10 days before the first immunization. Serum levels of ayw subtype–specific anti-HBsAg IgG (F) and HBsAg (G) (n = 3/group) were determined using ELISA. The detection limit in G is indicated by a dashed line. A total of 1 × 106 splenocytes from each mouse (n = 3/group) were collected on day 113. Specific B cell responses to HBsAg (subtype ayw) were assessed by a B cell ELISpot assay (H). (I and J) WT C57BL/6J mice (n = 5–6/group) were immunized s.c. with the mixture of aPD-L1–IFN-α (2 μg/mouse) and rHBsAg (1 μg/mouse) on days 0 and 14. For macrophage depletion in inguinal LNs, CLL (40 μL/mouse) was injected s.c. every 3 days, starting 7–10 days before the first immunization. HBsAg-specific IgM (I) and IgG (J) responses were measured on day 10 and day 21 after the first immunization, respectively. Data are shown as the mean + SEM (A, B, F, and G) or mean ± SEM (C–E and H–J) and are representative of at least 2 independent experiments. An unpaired 2-tailed Student’s t test was applied in C, D, and H–J. One-way ANOVA followed by Tukey’s test was applied in E. *P < 0.05; **P < 0.01; ****P < 0.0001. MΦ, macrophage; OD, optical density.