Virus-like Vesicles Expressing Multiple Antigens for Immunotherapy of Chronic Hepatitis B

Abstract

Infections with hepatitis B virus (HBV) can initiate chronic hepatitis and liver injury, causing more than 600,000 deaths each year worldwide. Current treatments for chronic hepatitis B are inadequate and leave an unmet need for immunotherapeutic approaches. We designed virus-like vesicles (VLV) as self-amplifying RNA replicons expressing three HBV antigens (polymerase, core, and middle surface) from a single vector (HBV-VLV) to break immune exhaustion despite persistent HBV replication. The HBV-VLV induces HBV-specific T cells in naive mice and renders them resistant to acute challenge with HBV. Using a chronic model of HBV infection, we demonstrate efficacy of HBV-VLV priming in combination with DNA booster immunization, as 40% of treated mice showed a decline of serum HBV surface antigen below the detection limit and marked reduction in liver HBV RNA accompanied by induction of HBsAg-specific CD8 T cells. These results warrant further evaluation of HBV-VLV for immunotherapy of chronic hepatitis B.

Keywords: Immunology; Medical Microbiology; Virology.

Graphical abstract

Figure 1

Virus-like Vesicle Platform for Therapeutic HBV Vaccine Expressing Polymerase (Pol), Ubiquitinated Core (Ub-HBc), and Middle Surface (MHBs) Antigens (A) Design of replicating VLV for expression of MHBs (MT2A), HBV Pol (PolT2A) single antigens, and for polycistronic expression of the three HBV antigens using different 2A self-cleaving peptides (3xT2A and Mix2A). (B) Validation of antigen expression in BHK-21 cells after infection with PolT2A or 3xT2A VLV (MOI = 1) by immunofluorescence at 20 h post infection using the antibodies specific for HBV Pol and HBcAg. (C) Validation of antigen expression in BHK-21 cells after infection with VLV expressing MHBs only (MT2A), Pol only (PolT2A), or the three antigens (3xT2A) by flow cytometry using antibodies specific for Pol, MHBs, HBcAg, and VSV-G. Geometric mean fluorescence intensity is shown for each antibody and VLV. (D) Evaluation of antigen expression and VLV replication in BHK-21 cells transfected with plasmid DNA for 3xT2A or Mix2A VLV using 2A-peptide specific antibody in western blots. Anti-VSV-G and anti-actin were also used as controls. The data are representative of three independent experiments.

Figure 2

Comparison of Immunogenicity of the 3xT2A and Mix2A VLV in Naive C57BL/6J Mice Accumulation of effector CD4⁺ and CD8⁺ T cells at day 7 after immunization with 3xT2A or Mix2A VLV. (A) Representative plots for CD62L and CD44 staining after gating on CD4⁺ or CD8⁺ T cells. (B) Absolute numbers of effector (CD44^hiCD62L^lo) T cells. (C) Frequency of HBV-specific IFNγ-producing CD8⁺ T cells identified after ex vivo stimulation with the indicated peptides from Pol (HBP-44, HBP-396, HBP-419), HBcAg (HBC-93), and surface (HBS-353 and HBS-371) quantified by intracellular staining and flow cytometry. (D) Numbers of IFNγ⁺ CD8⁺ T cells per spleen. (E) Frequency of HBV-specific double cytokine (TNF⁺IFNγ⁺)-producing CD8⁺ T cells, respectively. (F) Numbers of TNF⁺IFNγ⁺-double positive CD8⁺ T cells. Values in (C and E) are mean of 3–5 mice per group.

Figure 3

Efficacy of 3xT2A VLV as Prophylactic Vaccine in Acute HBV Infection CB6F1 mice were immunized with 3xT2A VLV or empty vector VLV for 6 weeks prior to challenge with AAV-HBV. (A) Levels of serum HBsAg (OD450) measured by specific ELISA from blood samples taken weekly. (B) Weekly levels of HBeAg. The values are mean ± SEM (n = 5). Asterisks indicate significant difference (*, p < 0.05, **, p < 0.01) in 3xT2A-immunized mice compared with PBS or empty vector VLV. (C) Abundance of HBV RNA in liver samples from mice euthanized on day 28 after challenge with AAV-HBV. TaqMan-based qRT-PCR was used to quantify HBV RNA and mouse GAPDH mRNA. The HBV RNA data were normalized to GAPDH and expressed as relative to the mean in the PBS group. Individual values and mean ± SD are shown. (D) Frequency of HBV-specific IFNγ⁺ producing cells was measured by ELISPOT on day 28 after challenge with AAV-HBV after ex vivo stimulation of splenocytes with the indicated peptides. Individual values and mean + SD are shown.

Figure 4

Effects of 3xT2A VLV on HBsAg levels in Chronic AAV-HBV Model Persistent HBV replication was established in C57BL/6 mice by delivery of HBV genome with AAV. The groups were balanced for HBsAg prior to immunization with empty vector VLV or 3xT2A at day 28 after AAV-HBV transduction. Booster DNA immunizations were carried out on days 65 and 84, as indicated by arrows, using a pool of 3 plasmids expressing MHBs, HBcAg, and HBV Pol for 3xT2A group or empty plasmid vectors for the empty vector VLV. (A–C) (A) Fold change in HBsAg levels (OD450). Values are mean ± SEM (n = 10 for days 28–70 and n = 5 thereafter). Quantification of HBsAg (ng/mL) after VLV prime at day 28 (B) and DNA boost at day 65 (C). Individual values and median are shown by symbols and bars, respectively. Asterisks in A indicate significant difference between 3xT2A and empty vector VLV-immunized animals (*, p < 0.05, **, p < 0.01).

Figure 5

Effects of 3xT2A VLV on Liver HBV RNA in Chronic AAV-HBV Model Liver samples were collected at day 70 or day 119 from the animals immunized as shown in Figure 4 for qRT-PCR analyses. (A) Abundance of HBV RNA was determined after normalization to mouse GAPDH mRNA and expressed relative to the mean of the empty vector VLV group (n = 5). Numbers of IFNγ⁺ CD8⁺ T cells per spleen at day 70 (B) and day 119 (C) were determined by intracellular flow cytometry after stimulation with HBV antigen peptides.