HBeAg seroconversion is associated with a more effective PD-L1 blockade during chronic hepatitis B infection

Abstract

Background & aims: Current therapies for chronic hepatitis B virus (HBV) infection control viral replication but do not eliminate the risk of progression to hepatocellular carcinoma. HBV-specific CD8 T cells are necessary for viral control, but they are rare and exhausted during chronic infection. Preclinical studies have shown that blockade of the PD-1:PD-L1 axis can restore HBV-specific T cell functionality. The aim of this study was to analyze how the clinical and treatment status of patients impacts the ability of HBV-specific T cells to respond to PD-L1 blockade.

Methods: Expression patterns of the PD-1:PD-L1/PD-L2 axis were analyzed in healthy donors and chronically infected patients in different clinical phases of disease. A functional assay was performed to quantify baseline HBV-specific T cell responses in chronically infected patients. Baseline responses were then compared to those attained in the presence of an anti-PD-L1 monoclonal antibody (MEDI2790).

Results: Chronically infected patients were characterized by the upregulation of PD-1 within the T cell compartment and a concomitant upregulation of PD-L1 on myeloid dendritic cells. The upregulation was maximal in HBV e antigen (HBeAg)-positive patients but persisted after HBeAg negativization and was not restored by long-term treatment. HBV reactivity, measured as frequency of HBV-specific T cells, was significantly higher in HBeAg-negative patients with lower HBV DNA levels, independently of HBV surface antigen or alanine aminotransferase levels. Anti-PD-L1 blockade with MEDI2790 increased both the number of IFN-γ-producing T cells and the amount of IFN-γ produced per cell in 97% of patients with detectable HBV reactivity, independently of patients' clinical or treatment status.

Conclusion: Patients with lower levels of HBV DNA and the absence of HBeAg have more intact HBV-specific T cell immunity and may benefit the most from PD-L1 blockade as a monotherapy.

Lay summary: Hepatitis B virus (HBV)-specific T cell responses during chronic infection are weak due to the upregulation of inhibitor molecules on the immune cells. In this study we show that the inhibitory PD-1:PD-L1 axis is upregulated during chronic HBV infection and successful antiretroviral therapy does not restore normal levels of PD-1 and PD-L1 expression. However, in HBV e antigen-negative patients, treatment with an anti-PD-L1 antibody can increase the functionality of HBV-specific T cell responses by an average of 2-fold and is a promising new therapy for patients with chronic HBV infection.

Keywords: Chronic HBV infection; HBV cure; HBV-specific T cells; MEDI2790; PD-1 blockade; PD-L1 blockade; checkpoint inhibitor; immunotherapy.

Graphical abstract

Fig. 1

Upregulation of the PD-1:PD-L1/PD-L2 axis in chronic HBV. (A) Representative flow plots for an HD and a chronic HBV-infected donor showing the PD-1 and PD-L1 expression in T cells, monocytes and mDCs (B) Pooled data showing the frequency of mDCs (left panel) and inflammatory monocytes (right panel) in HDs and individuals with chronic HBV infection. (C) Dysregulation in the PD-1:PD-L1/PD-L2 axis in CD8 T cells (upper panel) and mDCs (lower panel). SPICE data (left and middle panels) show the frequency of cells co-expressing 3 (PD-1⁺PD-L1⁺PD-L2⁺), 2, 1 or no markers (PD-1^-PD-L1^-PD-L2^-) at the same time. Arcs indicate cells expressing each individual marker. Pooled data (right panel) show the significant increase of PD-1-expressing CD8 T cells and PD-L1-expressing mDCs in chronic HBV infection. Mann-Whitney U test. *p ≪0.05, **p ≪0.001. HBV, hepatitis B virus; HD, healthy donor; mDCs, myeloid dendritic cells.

Fig. 2

Clinical correlates for the PD-1:PD-L1 axis dysregulation. Pooled data showing the frequency of (A) mDCs and inflammatory monocytes and (B) PD-1-expressing CD8 T cells and PD-L1-expressing mDCs in HDs, patients with chronic HBV infection under antiviral therapy, and untreated HBV-infected patients positive (eAg(+)) or negative (eAg(-)) for the HBeAg. Pooled data showing the frequency of PD-1-expressing CD8 T cells and PD-L1 expressing mDCs in HDs and chronically infected HBV patients with (C) different levels of HBsAg and (D) different levels of HBV DNA. Kruskal-Wallis test. *p ≪0.05, **p ≪0.001. HBeAg, HBV e antigen; HBsAg, HBV surface antigen; HBV, hepatitis B virus; HD, healthy donor; mDCs, myeloid dendritic cells.

Fig. 3

HBV-specific responses in chronic HBV infection. Pooled data showing (A) IFN-γ SFUs per million cells (left panel) and average intensity (right panel) and (B) Cytokine concentration in the assay supernatant after re-stimulation of the expanded PBMCs with either irrelevant peptide (actin) or HBV-capsid peptide pool. (C) IFN-γ SFUs per million cells (left panel) and spot intensity (right panel) after re-stimulation with HBV-capsid peptide pool in non-reactive (HBV-NR) and HBV-reactive (HBV-R+) patients. (D) Cytokine concentrations in the assay supernatant in non-reactive (HBV-NR) and HBV-reactive (HBV-R+) patients after re-stimulation. Mann-Whitney U test. **p ≪0.001, ***p ≪0.0001. HBV, hepatitis B virus; PBMCs, peripheral blood mononuclear cells; SFUs, spot-forming units.

Fig. 4

Clinical and biological correlates to HBV reactivity. (A) Frequency of HBV-reactive patients in patients positive or negative for HBeAg (left panel) and with low or high HBV DNA (right panel). Chi-square test. **p ≪0.001. (B) (Left panel) Frequency of HBV-reactive patients in patients negative for HBeAg with normal (Phase 3) or increased (Phase 4) ALT levels. (Right panel) IFN-γ SFUs per million cells among HBV-reactive patients, negative for HBsAg, with normal or altered ALT levels. Mann-Whitney U test. **p ≪0.001. Pooled data showing the frequency of (C) mDCs and inflammatory monocytes or (D) PD-L1 expression levels among these cell types in HDs, non-reactive (HBV-NR) and HBV-reactive (HBV-R+) patients. Mann-Whitney U test. *p ≪0.05, **p ≪0.001. ALT, alanine aminotransferase; HBeAg, HBV e antigen; HBV, hepatitis B virus; HD, healthy donor; mDCs, myeloid dendritic cells; SFUs, spot-forming units.

Fig. 5

PD-L1 blockade significantly increases HBV-specific responses. (A) Significant increase of both IFN-γ SFUs per million cells (left panel), IFN-γ SFUs per million cells fold-change (FC, middle panel) and spot intensity (right panel) after PD-L1 blockade with MEDI2790. Wilcoxon matched-pairs signed rank test. ***p ≪0.0001. (B) Cytokine concentrations in the assay supernatant in HBV-reactive patients in the presence or absence of an anti-PD-L1 blocking antibody. Wilcoxon matched-pairs signed rank test. ***p ≪0.0001. (C) Linear regression showing the correlation between the increase of IFN-γ SFUs per million cells (ΔSFU) in the presence of MEDI2790 and the frequency of PD-L1-expressing mDCs. Linear regression and 95% CI. p = 0.0577. HBV, hepatitis B virus; mDCs, myeloid dendritic cells; SFUs, spot-forming units.