PD-L1 Silencing in Liver Using siRNAs Enhances Efficacy of Therapeutic Vaccination for Chronic Hepatitis B

Abstract

In chronic hepatitis B virus (HBV) infection, virus-specific T cells are scarce and partially dysfunctional. Therapeutic vaccination is a promising strategy to induce and activate new virus-specific T cells. In long-term or high-level HBV carriers, however, therapeutic vaccination by itself may not suffice to cure HBV. One reason is the impairment of antiviral T cells by immune checkpoints. In this study, we used small-interfering RNA (siRNA) in combination with a heterologous prime-boost therapeutic vaccination scheme (TherVacB) to interfere with a major immune checkpoint, the interaction of programmed death protein-1 (PD-1) and its ligand (PDL-1). In mice persistently replicating HBV after infection with an adeno-associated virus harboring the HBV genome, siRNA targeting PD-L1 resulted in a higher functionality of HBV-specific CD8+ T cells after therapeutic vaccination, and allowed for a more sustained antiviral effect and control of HBV in peripheral blood and in the liver. The antiviral effect was more pronounced if PD-L1 was down-regulated during prime than during boost vaccination. Thus, targeting PD-L1 using siRNA is a promising approach to enhance the efficacy of therapeutic vaccination and finally cure HBV.

Keywords: HBV; PD-L1; RNAi; checkpoint inhibition; hepatitis; immunology; therapeutic vaccination.

Figure 1

Effect of PD-L1 checkpoint blockade on functionality of HBV-specific T cells. (A) C57Bl/6J mice (n = 5 per group) were infected intravenously with AAV-HBV resulting in persistent HBV replication. After nine weeks, mice were vaccinated two times using a combination of HBc and HBsAg adjuvanted with c-di-AMP (week 0 and week 2), and one time using an MVA expressing HBV core and S (week 4). Either 1 mg/kg siRNA or 100 µg antibody was administered i.v. one day prior to each vaccination. Mice were sacrificed at week 6. (B) HBV-specific (C₉₃) and MVA-specific (B8R) liver-associated lymphocytes (LAL) identified by multimer staining were co-stained for PD-1, TIM3 and LAG-3. The percentage of cells expressing one or more of these markers is indicated. (C–F) Liver-associated lymphocytes were isolated and restimulated for 16 h with peptide pools covering the HBV S- (S_pool) or core protein (C_pool) before intracellular cytokine staining of granzyme B (GzmB), IFNγ and TNFα was performed. (C) Representative flow cytometry stainings of LAL from single animals after stimulation with C_pool are shown. (D) Percentage of CD8⁺ LAL staining positive for the indicated cytokine per animal. Box plots show median, interquartile ratio (IQR) (box), and minimum to maximum (whiskers). (E,F) Five mice per group received either control or PD-L1-specific siRNA or antibody as indicated. LAL were isolated on day 14 after the last vaccination, stimulated with (E) S_pool or (F) C_pool and intracellular cytokine staining was performed. Mean ± SEM of the percentage of CD8⁺ T cells staining positive for the indicated cytokine are shown. Data from five animals per group are shown. Welch’s t-test or repeated measure two-way ANOVA with Tukey’s multiple comparison correction was performed; * = p < 0.05, ** = p < 0.01. *** = p < 0.001.

Figure 2

Antiviral efficacy of a combination of siPD-L1 and TherVacB. (A–E) Bl6 mice were infected intravenously with AAV-HBV resulting in persistent HBV replication. After nine weeks, mice were vaccinated two times using a combination of HBc and HBsAg adjuvanted with c-di-AMP (week 0 and week 2), and one time using an MVA expressing HBV core and S (week 4) (arrows in the graph indicate vaccinations); 1 mg/kg siRNA was administered i.v. one day prior to each vaccination. Mice were sacrificed at week 7 after start of vaccination. (A) HBsAg, (B), anti-HBs (C) HBeAg and (D) anti-HBe were measured in at indicated timepoints (arrows in the graph represent timepoints of vaccination). (E) Serum ALT activity was measured at the final timepoint. (F) Serum HBV DNA was measured using RT-qPCR at the final timepoint. (A–D) Graphs show mean and standard error of the mean (whiskers). Statistical analyses were performed using two-way ANOVA with Bonferroni correction for multiple comparison. (E) Box plots show median, interquartile ratio (IQR) (box), and minimum to maximum (whiskers). Data from six animals per group are shown. Repeated measure one-way ANOVA with Dunnett’s multiple comparison correction; * p < 0.05.

Figure 3

Effect of TherVacB and siPD-L1 on HBV persistence in the liver. (A) HBV pregenomic RNA (pgRNA) was quantified in liver lysates by RT-qPCR. (B,C) Quantification of hepatocytes that stained positive for HBc using immunohistochemistry. (B) One representative staining per group is shown. (C) Box plots show median, interquartile ratio (IQR) (box), and minimum to maximum (whiskers) numbers of cells with HBc⁺ cytoplasm per animal. (A,C) Data from four to six animals per group are shown. Kruskal–Wallis with Dunn’s multiple comparison correction; * p < 0.05, ** p < 0.01.

Figure 4

PD-L1 knock-down in the liver during prime, but not during boost vaccination enhances the effect of therapeutic vaccination. Bl6 mice (n = 3 to 4 per group) were infected intravenously with AAV-HBV to induce persistent HBV replication. After eight weeks, mice were vaccinated two times using a combination of HBc and HBsAg adjuvanted with c-di-AMP for prime vaccination (week 0 and week 2), and then boosted once using an MVA expressing HBV core and S (week 4); 1 mg/kg siRNA was administered i.v. one day prior to vaccination either twice before the prime vaccinations (siPrime) or once before the MVA boost (siBoost). Mice were sacrificed at week 10 after start of vaccination. Serum levels of HBsAg (A) and HBeAg (B) were measured at indicated timepoints. Dotted vertical lines indicate the three vaccinations. (C) Anti-HBs was quantified at the final timepoint. HBV pgRNA (D) in the liver and DNA (E) in the serum were quantified and measured by RT-qPCR. Repeated measure two-way ANOVA with Tukey´s multiple comparison correction; * p < 0.05.