Total antibody-positivity rates after the 1^st and 2^nd HBSS1 priming.

<p>A, alum; C, CpG; P, poly(I:C).</p

Vaccination groups and administration strategy.

<p>A, alum; C, CpG; P, poly(I:C).</p

Characterisation of recombinant adenovirus rAdSS1.

<p>(<b>A</b>) Schematic representation of recombinant adenovirus viral vectors encoding HBV S and PreS1 fusion genes. ITR, inverted terminal repeat. (B) Western blot detection of SS1 fusion protein expression in HEK293 cells infected with rAdSS1 using specific rabbit anti-PreS1 polyclonal antibodies. The bands of the expressed SS1 proteins are indicated by arrowheads.</p

In vitro cytokine production by splenocytes from immunized mice after rAdSS1 boosting.

<p>(A) IFN-γ; (B) IL-2. Supernatants were collected at 48h after HBV S and PreS1 peptide pool stimulation and cytokines were quantified by ELISA. IFN-γand IL-2 were detected and data were shown as means±SEMs. Statistical differences between groups were determined, and differences are shown as *p<0.05 and **p<0.01. SS1: HBSS1 A, alum; C, CpG; P, poly(I:C). Adjs: control group with adjuvants mixture.</p

Antibody responses in immunised mice.

<p>(<b>A</b>) Antisera were collected at 2 weeks after the 2^nd priming; (<b>B</b>) Antisera were collected at 2 weeks before rAdSS1 boosting; (<b>C</b>) Antisera were collected at 2 weeks after rAdSS1 boosting. Total IgG titres specific for the HBV antigen were determined by ELISA as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054126#s2" target="_blank">Methods</a> section. Statistical significance was analysed and shown as <i>**p</i><0.01 and <i>*p</i><0.05. SS1: HBSS1. A, alum; C, CpG; P, poly(I:C). Adjs: control group with adjuvants mixture.</p

Immunisation of C57BL/6 mice.

<p>Each group (12 mice/group) was primed twice with HBSS1 together with various adjuvant combinations at weeks 0 and 4. At week 14, all immunised groups were boosted with rAdSS1. Humoral and cellular immune responses were evaluated at the indicated times.</p

ELISpot analysis of IFN-γ secretion in mouse splenocytes.

<p>(A ) Splenocytes were isolated at weeks 6 after the 2nd priming; (B) Splenocytes were isolated at weeks 16 after the rAdSS1 boost. Data are expressed as spot-forming cell (SFC) responses to S and PreS1 peptide pools, and are presented as means with SEM. Significant <i>p</i> values between vaccinated groups are shown as * <i>p</i><0.05, and ** <i>p</i><0.01. SS1: HBSS1 A, alum; C, CpG; P, poly(I:C). Adjs: control group with adjuvants mixture.Data are shown as means ± SE or ± SEMs.</p

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.

<p>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, <i>P</i><0.05) and S- specific CD4 T cell responses for TNF-α(<i>P</i><0.05), mice receiving HBSS1 prime/RVJSS1 boost also generated markedly higher PreS1-specific CD4 T cell responses for TNF-α(<i>P</i><0.05).</p

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

<p>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.</p

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

<p>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 *<i>p</i><0.05.</p