Optimisation of prime-boost immunization in mice using novel protein-based and recombinant vaccinia (Tiantan)-based HBV vaccine

Abstract

Background: A therapeutic vaccine for chronic hepatitis B virus (HBV) infection that enhances virus-specific cellular immune responses is urgently needed. The "prime-boost" regimen is a widely used vaccine strategy against many persistence infections. However, few reports have addressed this strategy applying for HBV therapeutic vaccine development.

Methodology/principal findings: To develop an effective HBV therapeutic vaccine, we constructed a recombinant vaccinia virus (Tiantan) containing the S+PreS1 fusion antigen (RVJSS1) combined with the HBV particle-like subunit vaccine HBVSS1 to explore the most effective prime-boost regimen against HBV. The immune responses to different prime-boost regimens were assessed in C57BL/C mice by ELISA, ELISpot assay and Intracellular cytokine staining analysis. Among the combinations tested, an HBV protein particle vaccine priming and recombinant vaccinia virus boosting strategy accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres as well as the strongest multi-antigen (PreS1, and S)-specific cellular immune response. HBSS1 protein prime/RVJSS1 boost immunization was also generated more significant level of both CD4+ and CD8+ T cell responses for Th1 cytokines (TNF-α and IFN-γ).

Conclusions: The HBSS1 protein-vaccine prime plus RVJSS1 vector boost elicits specific antibody as well as CD4 and CD8 cells secreting Th1-like cytokines, and these immune responses may be important parameters for the future HBV therapeutic vaccines.

Figure 1. Characterization of recombinant vaccinia virus RVJSS1 and HBV particle-like subunit vaccine HBVSS1.

(A) Schematic diagram of recombinant vaccinia virus RVJSS1, which contains two expression cassettes in a back-to-back orientation, flanked by vaccinia virus TK region sequences. Virus RVJSS1 was a Tiantan strain vaccinia virus with a lacZ gene led by a p11 promoter inserted into the TK region; the expression cassette on the right consists of the SS1 fusion protein led by the 7.5 K promoter. (B) Immunofluorescence assay to confirm SS1 fusion protein expression. CEF cells were infected by the purified RVJSS1 virus and fixed, permeabilised, stained with rabbit anti-PreS1 antibody and fluorescein isothiocyanate (FITC) conjugated to a secondary antibody, and then visualised using fluorescence microscopy. (C) Western blot analyses to detect expression of SS1 fusion proteins in CEFs infected with RVJSS1 using specific antibodies. The expression bands of the SS1 proteins are indicated by arrowheads. (D) Negative staining of purified HBVSS1 particles vaccine using electron microscopy.

Figure 2. The anti-PreS1 and S antibody responses elicited by different regimens detected after first immunization (A–B) or second immunization(C–D).

Each group received a different prime–boost vaccination, and antiserum was collected 2 weeks after the each immunization. Total HBV antigen-specific IgG titres were determined by ELISA. The symbols represent the titers of the sera from the individual mice. The horizontal lines represent the means (n = 6). The statistic significance of the results was analyzed and indicated as *p<0.05.

Figure 3. Subtype analysis of the HBV antigen-specific IgGs in sera of mice immunised with different vaccine combinations.

Antigen specific IgG1 and IgG2a or IgG2b levels were determined using an IgG isotyping ELISA, as described in Materials and Methods. Sera were collected 2 weeks after the last immunization and diluted 1∶100. Bars indicate the average OD value at 450 nm (OD₄₅₀) of each group.

Figure 4. HBV S epitopes screening and ELISpot analysis of IFN-γ secretion in mouse splenocytes of each immunization group.

(A) HBV S epitopes were screened by IFN-γ ELISpot analysis. Actual sample wells of HBS-specific ASC spots. (B–D) HBV peptide-specific ASC frequency in each group. Data represent the average of spot-forming cells (SFCs) per million splenocytes from six mice/group plus the standard error. B:Splenocytes were collected 10 days after the first immunization and the number of IFN-γ secreting cells generated in response to S peptides; C–D: Splenocytes were collected 2 weeks after the last immunization and the number of IFN-γ secreting cells generated in response to PreS1 and S peptide pool stimuli respectively. Statistical differences between groups were determined, and differences are shown as *p<0.05 and **p<0.01. (E) Sample wells with spots with mock or HBV peptide stimulation.

Figure 5. ICS analyse of HBV PreS1- or S-specific CD4+ cells producing interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin-4 (IL-4) induced by heterologous rTTV and recombinant protein prime/boost immunization.

Splenocytes from four mice per group were isolated 14 days after last immunization. The splenocytes were exposed to HBV PreS1 peptide(S1–9) or S peptides pool and cytokine production was measured by monoclonal antibody staining and flow cytometric analysis. (a) Flow cytometer plot of results obtained from one representative individual mouse from each group. (b), Average (± SEM) of the percentage of IFN-γ-producing, TNF-α-producing and IL-4-producing CD4+ T cells obtained from four mice per group following stimulation with HBV PreS1 peptide (S1–9) or S peptides pool. Compared with RVJSS1 prime/HBSS1 boost, mice receiving HBSS1+Al(OH)3 prime/RVJSS1 boost generated markedly higher PreS1-specific CD4 T cell responses for two cytokines (IFN-γ and TNF-α, P<0.05) and S- specific CD4 T cell responses for TNF-α(P<0.05), mice receiving HBSS1 prime/RVJSS1 boost also generated markedly higher PreS1-specific CD4 T cell responses for TNF-α(P<0.05).

Figure 6. ICS analyse of HBV PreS1- or S-specific CD8+ cells producing interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin-4 (IL-4) induced by heterologous rTTV and recombinant protein prime/boost immunization.

Splenocytes from four mice per group were isolated 14 days after last immunization. The splenocytes were exposed to HBV PreS1 peptide(S1–9) or S peptides pool and cytokine production was measured by monoclonal antibody staining and flow cytometric analysis. (a) Flow cytometer plot of results obtained from one representative individual mouse from each group. (b), Average (± SEM) of the percentage of IFN-γ-producing, TNF-α-producing and IL-4-producing CD8+ T cells obtained from four mice per group following stimulation with HBV PreS1 peptide (S1–9) or S peptides pool. Compared with RVJSS1 prime/HBSS1 boost, mice receiving HBSS1+Al(OH)3 prime/RVJSS1 boost generated markedly higher PreS1-specific CD8 T cell responses for IFN-γ(P<0.05) and S- specific CD8 T cell responses for TNF-α(P<0.05), mice receiving HBSS1 prime/RVJSS1 boost generated markedly higher PreS1- and S-specific CD8 T cell responses for TNF-α(P<0.05).