A Highly Attenuated Vesicular Stomatitis Virus-Based Vaccine Platform Controls Hepatitis B Virus Replication in Mouse Models of Hepatitis B

Abstract

Therapeutic vaccines may be an important component of a treatment regimen for curing chronic hepatitis B virus (HBV) infection. We previously demonstrated that recombinant wild-type vesicular stomatitis virus (VSV) expressing the HBV middle surface glycoprotein (MHBs) elicits functional immune responses in mouse models of HBV replication. However, VSV has some undesirable pathogenic properties, and the use of this platform in humans requires further viral attenuation. We therefore generated a highly attenuated VSV that expresses MHBs and contains two attenuating mutations. This vector was evaluated for immunogenicity, pathogenesis, and anti-HBV function in mice. Compared to wild-type VSV, the highly attenuated virus displayed markedly reduced pathogenesis but induced similar MHBs-specific CD8⁺ T cell and antibody responses. The CD8⁺ T cell responses elicited by this vector in naive mice prevented HBV replication in animals that were later challenged by hydrodynamic injection or transduction with adeno-associated virus encoding the HBV genome (AAV-HBV). In mice in which persistent HBV replication was first established by AAV-HBV transduction, subsequent immunization with the attenuated VSV induced MHBs-specific CD8⁺ T cell responses that corresponded with reductions in serum and liver HBV antigens and nucleic acids. HBV control was associated with an increase in the frequency of intrahepatic HBV-specific CD8⁺ T cells and a transient elevation in serum alanine aminotransferase activity. The ability of VSV to induce a robust multispecific T cell response that controls HBV replication combined with the improved safety profile of the highly attenuated vector suggests that this platform offers a new approach for HBV therapeutic vaccination.IMPORTANCE A curative treatment for chronic hepatitis B must eliminate the virus from the liver, but current antiviral therapies typically fail to do so. Immune-mediated resolution of infection occurs in a small fraction of chronic HBV patients, which suggests the potential efficacy of therapeutic strategies that boost the patient's own immune response to the virus. We modified a safe form of VSV to express an immunogenic HBV protein and evaluated the efficacy of this vector in the prevention and treatment of HBV infection in mouse models. Our results show that this vector elicits HBV-specific immune responses that prevent the establishment of HBV infection and reduce viral proteins in the serum and viral DNA/RNA in the liver of mice with persistent HBV replication. These findings suggest that highly attenuated and safe virus-based vaccine platforms have the potential to be utilized for the development of an effective therapeutic vaccine against chronic HBV infection.

Keywords: adeno-associated virus; chronic hepatitis B; immunotherapy; immunotolerance; therapeutic vaccines; vesicular stomatitis virus.

FIG 1

Compared to VSV-MHBs, N4CT1-MHBs displays a low replication rate and reduced cytopathic effects in vitro. (A) VSV vectors used for immunizations. (B) BHK cells were infected with the indicated viruses or not infected, and cell lysates were collected at 12 h postinfection and assayed for MHBs or VSV protein (M, N, and G) expression by Western blotting. (C) BHK cells in triplicate wells were infected with the indicated viruses, and the titers in medium were determined to compare the replication of both viruses. Error bars denote standard errors of the means (SEM). (D) Plaque size comparison in infected BHK cells.

FIG 2

Despite differences in pathogenicity, N4CT1-MHBs and VSV-MHBs generate similar immune responses in naive mice. (A) Daily weight changes of CB6F1 mice infected intranasally with either N4CT1-MHBs or VSV-MHBs (1 × 10⁶ PFU). (B) Ag-specific CD8⁺ T cells measured by an IFN-γ ELISPOT assay in the spleens of CB6F1 mice immunized with the vectors (1 × 10⁶ PFU, i.m.) or PBS on day 7 postimmunization (n = 5 mice/group). (C) Anti-HBs antibody measured by an ELISA in CB6F1 mouse serum on week 8 postimmunization (n = 5 to 6 mice/group). (D) Ag-specific CD8⁺ T cells measured by an IFN-γ ELISPOT assay in the spleens of DO mice at 2 weeks postimmunization (n = 6 to 8 mice/group). Error bars denote SEM.

FIG 3

Immunization with N4CT1-MHBs protects mice against HBV hydrodynamic challenge. CB6F1 mice were immunized intramuscularly with 1 × 10⁶ PFU of VSV-MHBs or N4CT1-MHBs and challenged 6 weeks later by hydrodynamic injection (HDI) of the pHBV1.3 plasmid. (A) HBeAg levels measured by an ELISA relative to those in the unimmunized group at day 1 (mean concentration, 307 ± 179 ng/ml) in sera collected on the indicated days postchallenge. (B) Hepatic HBV DNA and RNA on day 7 postchallenge quantified by qPCR and RT-qPCR, respectively, shown as changes in threshold cycle values. (C) Relative liver CD4 and CD8 mRNA quantified by RT-qPCR. (D) HBV-specific CD8⁺ T cell response in the spleen on day 7 postchallenge measured by an IFN-γ ELISPOT assay (n = 2 to 4 mice/group). Error bars denote SEM.

FIG 4

Immunization with N4CT1-MHBs elicits Ag-specific immune responses that prevent persistent HBV replication in mice. CB6F1 mice were immunized with the indicated vectors (1 × 10⁶ PFU, i.m.) and challenged i.v. with 1 × 10¹¹ genome copies of AAV-HBV at week 6 postimmunization. (A) Serum HBsAg levels measured by ELISAs relative to those in the week 3 postchallenge PBS group (mean concentration, 48 ± 23 ng/ml). (B) Anti-HBs quantification by an ELISA in serum prior to challenge (pre) and at week 3 (W3) and week 4 postchallenge. (C) Serum HBeAg levels measured by an ELISA relative to those in the week 3 postchallenge PBS group (mean concentration, 79 ± 7 ng/ml). (D and E) Hepatic HBV RNA and genomic DNA (gDNA) levels on week 4 postchallenge measured by RT-qPCR and qPCR, respectively. C_T, threshold cycle. (F) MHBs-specific CD8⁺ T cell detection by an IFN-γ ELISPOT assay in the spleen on week 4 postchallenge (n = 5 to 6 mice/group). Error bars denote SEM.

FIG 5

Immunization with N4CT1-MHBs overcomes immune tolerance in HBV^tg mice. (A) Measurement of serum HBeAg (left) and HBsAg (right) in HBV.CB6F1 mice by an ELISA. (B) HBV-specific CD8⁺ T cells in the spleen at week 2 postimmunization. (C) Anti-HBs Ab levels in the serum before immunization (pre) and at week 2 postimmunization. The assay limit of detection is shown by a dotted line (n = 9 mice/group). Error bars denote SEM.

FIG 6

N4CT1-MHBs treatment of mice with persistent HBV replication results in significant HBV Ag reduction and Ag-specific CD8⁺ T cell induction. C57BL/6 mice received 1 × 10¹¹ genome copies of AAV-HBV i.v. and on week 8 posttransduction were treated with the indicated vectors (1 × 10⁶ PFU, i.m.) or PBS. (A and B) Serum HBV Ag levels at different time points measured by an ELISA relative to those on week 8 after AAV-HBV transduction. The mean concentrations ± SEM of serum HBsAg and HBeAg at the time of treatment were 182 ± 11 and 94 ± 9 ng/ml, respectively. (C) IFN-γ ELISPOT analysis of MHBs-specific CD8⁺ T cell responses in the spleen on week 3 posttreatment. (D) Serum HBsAg levels at different time points measured by an ELISA relative to those at week 8. The mean concentration of HBsAg at the time of treatment was 296 ± 36 ng/ml (n = 5 to 9 mice/group). Error bars denote SEM.

FIG 7

A single treatment by N4CT1-MHBs in mice with persistent HBV replication results in viral Ag clearance from serum, reduced viral replication in the liver, transient ALT elevation, and increased Ag-specific CD8⁺ T cell frequency. C57BL/6 mice received 1 × 10¹¹ genome copies of AAV-HBV i.v. and on week 8 posttransduction were treated with the indicated vectors (1 × 10⁶ PFU i.m.). (A and B) Serum viral Ag levels at different time points measured by an ELISA relative to those on week 8 after AAV-HBV transduction. The concentrations ± SEM of serum HBsAg and HBeAg at the time of treatment were 166 ± 17 and 106 ± 7 ng/ml, respectively. (C) IFN-γ ELISPOT analysis of MHBs-specific CD8⁺ T cells in the spleens of mice at different weeks posttreatment. (D) Serum ALT activity before treatment and at the indicated weeks posttreatment. (E) Liver CD8 and CD4 mRNA expression measured by RT-qPCR. (F) Relative hepatic HBV RNA (normalized to GAPDH) and HBV genomic DNA (in 50 mg of liver tissue) quantified by RT-qPCR and qPCR, respectively. Background PCR amplification (limit of detection) in nontransduced mouse livers is shown by a dotted line. Data shown in panels A, B, and D are derived from two groups of mice that were euthanized on week 5 posttreatment, and data shown in panels C and E to G are from two groups of mice euthanized at the indicated time points (n = 5 mice/group). Error bars denote SEM.

FIG 8

Increased frequency of HBV-specific CD8⁺ T cells in the livers of mice treated with N4CT1-MHBs. AAV-HBV-transduced C57BL/6 mice with persistent HBV replication were treated with N4CT1-MHBs (1 × 10⁶ PFU i.m.) or PBS. IHL were isolated at week 3 posttreatment, stimulated overnight in the presence or absence of HBV peptides, and stained for expression of CD8 and IFN-γ. (A) Representative flow cytometry plots of IHL. SSC, side scatter. (B) Frequency of CD8⁺ T cells among IHL. (C) Frequency of HBV-specific CD8⁺ IFN-γ⁺ cells among IHL after in vitro stimulation with HBV peptides.