A vesicular stomatitis virus-based hepatitis B virus vaccine vector provides protection against challenge in a single dose

Abstract

As one of the world's most common infectious diseases, hepatitis B virus (HBV) is a serious worldwide public health problem, with HBV-associated liver disease accounting for more than half a million deaths each year. Although there is an effective prophylactic vaccine currently available to prevent infection, it has a number of characteristics that are suboptimal: multiple doses are needed to induce long-lasting immunity, immunity declines over time, it does not elicit protection in some individuals, and it is not effective therapeutically. We produced a recombinant vesicular stomatitis virus (VSV)-based vaccine vector expressing the HBV middle envelope surface protein (MS) and found that this vector was able to efficiently generate a strong HBs-specific antibody response following a single immunization in mice. A single immunization with the VSV-MS vector also induced robust CD8 T-cell activation. The CD8 T-cell response was greater in magnitude and broader in specificity than the response generated by a vaccinia virus-based vaccine vector or by recombinant protein immunization. Furthermore, a single VSV-MS immunization provided protection against virus challenge in mice. Given the similar antibody titers and superior T-cell responses elicited from a single immunization, a VSV-based HBV vaccine may have advantages over the current recombinant protein vaccine.

FIG. 1.

Characterization of MS expression from recombinant VSV. (A) Gene encoding HBV middle envelope (MS) inserted in the fifth position of the VSV genome, diagrammed from the 3′-to-5′ orientation of the negative-stranded viral RNA genome. (B) BHK cells were harvested 6 h postinfection with VSV or VSV-MS and assayed for MS expression by Western blotting (WB) with anti-HBsAg monoclonal antibody. Uninf, uninfected. (C) Alternatively, 6 h postinfection, BHK cells were fixed with 2% paraformaldehyde prior to permeabilization and staining. MS localization was detected using primary mouse anti-HBsAg (α-HBS) and secondary Alexa Fluor 488-labeled goat anti-mouse antibodies. α-VSV-G, anti-VSV-G. (D) Medium from VSV-MS-infected BHK cells was layered over a continuous sucrose gradient (5 to 25%) and centrifuged at 30,000 × g for 20 min. Gradient fractions were collected, and migration densities were determined for HBsAg by ELISA and VSV-M protein by WB quantification using polyclonal anti-VSV antibody. O.D., optical density.

FIG. 2.

Specific CD8⁺ T-cell responses are elicited following a single immunization with VSV-MS. CB6F1 mice were immunized with DMEM (n = 4), control VSV vectors (n = 5), or VSV-MS (n = 5). (A) Seven days postimmunization, splenocytes were harvested and analyzed using an IFN-γ ELISPOT assay. The number of cells responding to stimulation with HBV S- or VSV-specific peptides is represented as quantification of the number of spot-forming cells (SFC) per 10⁶ splenocytes. (B) A peptide library containing 76 15-mer peptides spanning the HBV envelope was pooled into 9 groups of 8 peptides and 1 group of 4 peptides and used in an IFN-γ ELISPOT assay conducted 7 days postimmunization with VSV-MS (n = 3). (C) The 9 peptides from responsive pools E and I were then tested individually in an IFN-γ ELISPOT assay to determine the immunogenic epitopes. All error bars represent standard error (SE).

FIG. 3.

VSV-MS elicits stronger cellular and humoral immune responses than recombinant MS protein and Engerix-B vaccination. (A) CB6F1 mice were immunized with VSV-MS (i.m., n = 5; i.n., n = 5), recombinant S protein (rHBsAg) (n = 5), or Engerix-B (n = 5). Seven days postimmunization, splenocytes were isolated and analyzed for CD8, CD3, and IFN-γ expression by flow cytometry. The values shown represent CD8⁺ and CD3⁺ gated cells. (B) CB6F1 mice were immunized with VSV-MS (i.m., n = 5; i.n., n = 5), recombinant S protein (rHBsAg) (n = 5), or Engerix-B (n = 10). Quantitative HBsAb ELISAs were conducted on serum collected 4 weeks postimmunization to determine specific antibody titers. All values are presented as the average from each group; error bars represent SE.

FIG. 4.

VSV-MS generates a CD8⁺ T-cell response superior to that of VV-S. CB6F1 mice were immunized with DMEM (n = 2), VSV (n = 3), VSV-MS (n = 7), or VV-S (n = 6). (A) An IFN-γ ELISPOT assay was conducted 7 days postimmunization using four HBV S-specific peptides and two VSV-specific peptides. (B) Three weeks after a DNA-MS prime, CB6F1 mice were boosted with DMEM (n = 3), DNA-MS (n = 3), VV-S (n = 3), and VSV-MS (n = 4). Seven days postimmunization, splenocytes were isolated and analyzed for CD8, IFN-γ, TNF-α, and IL-2 expression by flow cytometry. CD8⁺ T cells were gated, and IFN-γ-producing cells are presented as the percent average from each group. Error bars represent SE. (C) Boolean gating analysis was used to identify sets of CD8⁺ T cells expressing each possible combination of cytokines (IFN-γ, TNF-α, and IL-2). Data are presented as the average from each group with triple-, double-, and single-cytokine-producing cells represented as the percentage of total cytokine-producing CD8⁺ T cells.

FIG. 5.

VSV-MS provides protection against vaccinia virus challenge. C57BL/6 mice were primed 60 days prior to heterologous challenge. The following prime/challenge strategies were used: VSV/VV-S (n = 4), VSV-MS/VV-S (n = 4), and VSV-MS/VV (n = 2). (A) Body weight was measured for 14 days following challenge. **, P < 0.002; and *, P < 0.02 (weight loss in VSV-MS-immunized/VV-S-challenged mice compared to that in VSV-immunized/VV-S-challenged mice). (B) Splenocytes were harvested 14 days postchallenge and stimulated using HBV S- and VSV-specific peptides in an ELISPOT assay to measure recall responses. All values are presented as the average from each group, and error bars indicate the SE.

FIG. 6.

VSV-MS provides protection against hydrodynamic HBV challenge. CB6F1 mice were immunized with VSV-MS (n = 8) or empty VSV (n = 7). Sixty days postimmunization, HBV replication was induced by hydrodynamic injection of HBV 1.3 plasmid (10 μg), and analysis was performed on days 4 (n = 10) and 7 (n = 5) postchallenge. (A) Liver-associated HBV DNA and RNA were measured using Southern blot (SB) and Northern blot (NB) analyses, respectively. RC, relaxed circular DNA; SS, single-stranded DNA. (B) HBcAg-positive hepatocytes per 10× field were quantified after liver sections were stained with anti-HBcAg antibody. Values represent the average of two fields per sample with SE. Serum analysis for HBeAg (C) and HBsAg (D) was completed by ELISA on days 0, 1, 4, and 7 post-hydrodynamic challenge. O.D., optical density. (E) HBsAb titers were determined using a quantitative ELISA, and (F) serum alanine aminotransferase (sALT) activity was measured using Infinity ALT reagent. All values are presented as the average from each group, and error bars represent SE.

FIG. 7.

VSV-MS-mediated protection correlates with CD8 T-cell recall response. CB6F1 mice were immunized with VSV-MS (n = 5) or recombinant MS protein (rHBsAg) (n = 4); a control group of unimmunized (Unimm.) mice was also included (n = 4). Sixty days postimmunization, HBV replication was induced by hydrodynamic injection of HBV 1.3 plasmid (10 μg), and analysis was performed on day 7 postchallenge. (A) Serum analysis for HBeAg was completed by ELISA on days 0, 1, 4, and 7 post-hydrodynamic challenge. (B) Splenocytes and intrahepatic lymphocytes (IHL) were isolated and analyzed for CD8, CD3, and IFN-γ expression by flow cytometry. CD8⁺ CD3⁺ T cells were gated, and IFN-γ-producing cells are presented as the percent average from each group for unpooled samples; error bars represent SE. (C) Liver-associated HBV DNA was measured using Southern blot analysis. RC, relaxed circular DNA; SS, single-stranded DNA. (D) CD8 T cells were depleted by anti-CD8 (αCD8) antibody injection 1 day prior to hydrodynamic challenge and again on day 2 postchallenge, and analysis was performed on day 7 postchallenge. Liver-associated HBV DNA was measured using Southern blot analysis. IHL were pooled and analyzed for CD8⁺ CD3⁺ T cells by flow cytometry.