An autophagosome-based therapeutic vaccine for HBV infection: a preclinical evaluation

Abstract

Background: For more than 240 million chronic HBV carriers worldwide, effective therapeutic HBV vaccines are urgently needed. Recently, we demonstrated that autophagosomes were efficient antigens carriers and capable to cross-prime robust T-cell responses and mediate regression of multiple established tumors. Here we tested whether autophagosomes derived from HBV expressing cells could also function as a therapeutic vaccine.

Methods: We generated an autophagosome-based HBV vaccine from HBV-expressing hepatoma cells and examined its ability to induce polyvalent anti-HBV T-cell responses and therapeutic efficacy in mouse models that mimic acute and chronic HBV infection in human.

Results: When compared to the vaccine based on recombinant HBsAg, autophagosome-based HBV vaccine cross-primed multi-specific anti-HBV T-cell responses and significantly reduced HBV replication and HBcAg expression in livers of both acute and chronic mouse models. Therapeutic effect of this HBV vaccine depended on anti-HBV CD8(+) effector T cells and associated with increased HBsAg and HBcAg specific IFN-γ producing T cells in the chronic mouse model.

Conclusions: These results indicated that autophagosome-based HBV vaccine could effectively suppress the HBV replication, clear the HBV infected hepatocytes, and break the HBV tolerance in mouse model. The potential clinical application of autophagosome-based HBV vaccine is discussed.

Figures

Figure 1

Detection of HBV antigens in DRibbles. HepG2.2.15 cells were treated without (lane 1) or with Bortizomib alone (lane 2), Rapamycin alone (lane 3), Bortizomib plus Rapamycin (lane 4) or combination of Bortizomib, Rapamycin and NH4Cl (lane 5) for 18 h. Lysates were prepared for the HBsAg and the LC3 conversion analysis by western blot (A,B). The HBV+ DRibbles prepared from both untreated and treated cells were analyzed for LC-3-I and LC-3-II by western blot analysis (C). Anti-β-actin antibody was included as a sample loading control. Morphology of DRibbles was observed by transmission electron microscopy (D). The amount of HBeAg (E) and HBsAg (F) in the DRibbles were quantified by ELISA.

Figure 2

HBV+DRibbles elicited HBV-specific protective immune response. The schematic diagram outlines the experiment protocol (A). The optimal dose of HBV+ DRibbles was determined by ELISPOT assay (n = 3). Lymphocytes from mice received HBV+ DRibbles generated a high number of IFN-γ secreting cells in 2 × 105 lymphocytes (B,C). Vaccinated mice were injected hydrodynamically with 10 μg of HBV plasmid DNA (n = 5) 7 days later. The serum HBV DNA (D) and HBeAg (E) was determined by real-time PCR and ELISA. Intrahepatic HBcAg was visualized by immunohistochemical staining (×200) (F). Results represent three independent experiments.

Figure 3

CD8+T cells mediated protection induced by HBV+DRibbles. The schematic diagram outlines the experiment protocol (A). Mice were injected hydrodynamically with HBV plasmid DNA 7 days after vaccination and immediately subjected to intraperitoneal injection of PBS, anti-CD4 mAb, anti-CD8 mAb or anti-CD4 mAb plus anti-CD8 mAb, respectively (n = 5) (B-D). The serum HBeAg (B) and HBV DNA (C) were detected by ELISA and real-time PCR. Intrahepatic HBcAg was visualized by immunohistochemistry (×200) (D). Lymphocytes from HBV+ DRibbles vaccinated mice (n = 3) were co-incubated with target cells (the effector to target ratio was 100:1) before the supernatant IFN-γ (E) and AST (F) were assayed by ELISA and by clinical chemistry analyzer. These experiments were repeated three times with comparable results.

Figure 4

Therapeutic efficacy of HBV+DRibbles vaccine. The schematic diagram outlines the experiment protocol (A). Lymphocytes from the acute HBV-infected mice received HBV+ DRibbles or HBsAg or PBS were isolated and re-stimulated with HBV antigens or peptides and the number of IFN-γ producing cells in 2 × 105 lymphocytes was detected as described (n = 3) (B-D). Serum HBeAg (E), HBsAg (F), HBV DNA copy number (G) and the percentage of HBcAg+ hepatocytes (H) of immunized mice or control mice were measured as mentioned above (n = 6). These experiments were repeated three times.

Figure 5

Immunized lymphocytes transfer adoptive immunity against HBV. The schematic diagram outlines the experiment protocol (A). HBV+ DRibbles elicited IFN-γ producing cells in 2 × 105 lymphocytes against multiple HBV antigens or peptides (n = 3) (B,C). A total of 1 × 108 lymphocytes were adoptively transferred intravenously into each of the new ‘HBV tolerant’ mouse. At day 7, 14 and 21 after adoptive transfer, serum samples were collected for detection of HBsAg (D), HBV DNA copy number (E). The liver tissues were harvested on day 21 and HBcAg+ hepatocytes were determined as described (n = 4) (F). These experiments were repeated three times with comparable results.