圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) CA, USA)was added and the reactions were terminated by the addition of 50 ul/well 2M H2S04. The absorbance at 450nm was immediately read on a microplate reader(Molecular devices, Sunnyvale, CA, USA). A standard curve of IgG standard was used to transform the absorbance to antibody concentration Flow cytometry For lymphocytes differentiation, blood samples were harvested at indicated time points and monocytes were separated though isodensity centrifugation. Thereafter, approximately 10 lls were re suspended in 100 uL of PBS and incubated with 2 ug anti-CD3c FITC together with anti-CD4 PE or anti-CD8 PE antibodies(all from BioLegend, San Diego, CA, USA)for 20 min on ice. Thereafter. the cells were washed twice with pbs and fle formed using a FACSort instrument(BD Biosciences, San Jose, CA, USA). Data was analyze ng FlowJo software(Tree Star, San Caros, CA, USA) Statistical analysis The statistical significance of the results was determined using the Provantis system(SAS, US) The homogeneity of variance was examined with Levene's test A one-way aNOVa was used to determine the significance of the data(P>0.05). For groups in which P <0.05, Dunnetts test was used for comparative analysis. For groups with heterogeneity of variance, Levene's test was used after log transforming the data. The Kruskal-Wallis test was used for data that still had heterogeneity of variance after log transformation. When evaluating differences between experimental and control samples, P<0.05 was considered to be statistically significant. Results SBP adjuvant enhances HBsAg-specific antibody production in rats The serum antibody titer correlates with the protection offered by vaccines. To determine the ffects of SBP on the immunogenicity of HBV vaccine, HBs Ag-specific antibody was assessed by enzyme-linked immunosorbent assay in rats that received various dosages of HBs Ag with or without the SBP adjuvant. Higher dosages of HBs Ag adjuvanted with SBP triggered more Igg generation in rats after day 14, and IgG production doubled on day 21 compared with the non-adjuvanted group(Fig 2). For the lowest dosage group, an increase in IgG was first detected on day 21, and the rats that received SBP had higher levels of IgG production. The results also show that SBP plays a role in the maintenance of antibody levels over time. In the HBsAg dose: 10ug HBsAg dose: 2. 5ug HBsAg dose: 1. 25ug Fig 2. HBsAg specific antibody induced by sBP adjuvanted and non-adjuvanted es. rats were collected on indicated time points and anti-HBs total lgG was measured by ELISA Results ale.Nether were immunized intramuscularly 3 times at a 2-week interval with vaccine alone(H)or tog with SBP(H-S). There are 6 groups with 3 injected HBsAg dose: 1.25ug, 2.5ug and 10ug. Blood sar doi: 10.1371/journal pone. 0170313. g002 PLOS ONE DO1: 10. 1371/journal pone. 0170313 January 4/1CA, USA) was added and the reactions were terminated by the addition of 50 μl/well 2M H2SO4. The absorbance at 450nm was immediately read on a microplate reader (Molecular devices, Sunnyvale, CA, USA). A standard curve of IgG standard was used to transform the absorbance to antibody concentration. Flow cytometry For lymphocytes differentiation, blood samples were harvested at indicated time points and monocytes were separated though isodensity centrifugation. Thereafter, approximately 106 cells were re suspended in 100 μL of PBS and incubated with 2 μg anti-CD3c FITC together with anti-CD4 PE or anti-CD8 PE antibodies (all from BioLegend, San Diego, CA, USA) for 20 min on ice. Thereafter, the cells were washed twice with PBS and flow cytometry was per￾formed using a FACSort instrument (BD Biosciences, San Jose, CA, USA). Data was analyzed using FlowJo software (Tree Star, San Caros, CA, USA). Statistical analysis The statistical significance of the results was determined using the Provantis system (SAS, US). The homogeneity of variance was examined with Levene’s test. A one-way ANOVA was used to determine the significance of the data (P > 0.05). For groups in which P  0.05, Dunnett’s test was used for comparative analysis. For groups with heterogeneity of variance, Levene’s test was used after log transforming the data. The Kruskal-Wallis test was used for data that still had heterogeneity of variance after log transformation. When evaluating differences between experimental and control samples, P  0.05 was considered to be statistically significant. Results SBP adjuvant enhances HBsAg-specific antibody production in rats The serum antibody titer correlates with the protection offered by vaccines. To determine the effects of SBP on the immunogenicity of HBV vaccine, HBsAg-specific antibody was assessed by enzyme-linked immunosorbent assay in rats that received various dosages of HBsAg with or without the SBP adjuvant. Higher dosages of HBsAg adjuvanted with SBP triggered more IgG generation in rats after day 14, and IgG production doubled on day 21 compared with the non-adjuvanted group (Fig 2). For the lowest dosage group, an increase in IgG was first detected on day 21, and the rats that received SBP had higher levels of IgG production. The results also show that SBP plays a role in the maintenance of antibody levels over time. In the Fig 2. HBsAg specific antibody induced by SBP adjuvanted and non-adjuvanted vaccines. Rats (n = 10/group) were immunized intramuscularly 3 times at a 2-week interval with vaccine alone (H) or together with SBP (H-S). There are 6 groups with 3 injected HBsAg dose: 1.25μg, 2.5μg and 10μg. Blood samples were collected on indicated time points and anti-HBs total IgG was measured by ELISA. Results are expressed as the means ± SEM, * p<0.05. doi:10.1371/journal.pone.0170313.g002 A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 4 / 11