圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) B Eo 31d37d 0d8d14d20d31d37d Fig 5 SBP adjuvanted hepatitis B vaccine could induce higher antibody titers and higher percentage of ells Macaca fascicularis were immunized intramuscularly at days 1, 9, 15, 32 with vaccine alone(H)or together SBP(H-S).(A)Blood samples were collected on indicated time points and HBsAg specific antibody titers were easured. (B)Percentage of CD4+ T cells was also measured at these time points. Results are expressed as the means±SEM,p<0 doi: 10.1371/journal pone. 0170313.g005 T cell-dependent immune responses were also important to antibody production. Our results showed that the animals who received the SBP-adjuvanted vaccine had slightly elevated levels of CD4+ T cells, which suggests that SBP may promote the differentiation and matura tion of T cells. However, the levels of T cells, and other hematological parameters, were within the normal range and the differences between the groups was not statistically significant( data Discussion Although the existing HBV vaccine provides many people with significant protection against infection [13], a more effective vaccine is required for poor-response groups [14, 15]. In a pre- vious study, we found an HBsAg-binding protein that could promote the immune process both in vitro and in vivo. Its adjuvanticity in connection with several ar [16], and rotavirus [1Z)) has been evaluated in mice and the results indicate that it has the otential to be a new adjuvant for use in human vaccines During vaccine and adjuvant development, various pre-clinical testing is required before linical safety evaluation [12, 18]. It is very important to evaluate potential side effects via toxi ology tests in animals. Standard GLP non-clinical pharmacology, toxicity, and allergy studies The results demonstrate that SBP-adjuvanted HBv m pleted in rodents in the present study for SBP and SBP-adjuvanted HBV vaccine were cor vaccine could enhance the production of anti-HBsAg antibody significantly. Throughout both acute(single dosage)and sub-acute (repeated dosage)studies, SBP and SBP-adjuvanted vaccine were well-tolerated. No trea ment-related effects in food consumption, body weight, clinical signs, body temperature, opl thalmic testing, or organ-body weight ratios were noted. White nodules were found at the injection site in the repeated-dosage vaccine group 3 days sis,mesenchyme inflammatory cell infiltration, macrophage aggregation, and abscesses. Mapi after the final injection. Histopathological examination of the nodules showed myofiber necro mechanical stimulation, including myofiber necrosis and mesenchyme inflammatory cell infil tration, was also observed in SBP group Injection site reactions such as these are not uncom- mon with adjuvanted vaccines: Lawrence Segal et al. reported that human papillomavirus vaccine could induce injection site reactions in rabbits and rats [18], and Penina Haber et al. showed that inactivated influenza vaccine induced injection site reactions in pre-licensure clinical trials [19, 20]. Some statistically significant changes in blood biochemistry parameters PLOS ONE DO1: 10. 1371/journal pone. 0170313 January 19, 2017T cell—dependent immune responses were also important to antibody production. Our results showed that the animals who received the SBP-adjuvanted vaccine had slightly elevated levels of CD4+ T cells, which suggests that SBP may promote the differentiation and matura￾tion of T cells. However, the levels of T cells, and other hematological parameters, were within the normal range and the differences between the groups was not statistically significant (data not shown). Discussion Although the existing HBV vaccine provides many people with significant protection against infection [13], a more effective vaccine is required for poor-response groups [14, 15]. In a pre￾vious study, we found an HBsAg-binding protein that could promote the immune process both in vitro and in vivo. Its adjuvanticity in connection with several antigens (HBsAg, rabies [16], and rotavirus [17]) has been evaluated in mice and the results indicate that it has the potential to be a new adjuvant for use in human vaccines. During vaccine and adjuvant development, various pre-clinical testing is required before clinical safety evaluation [12, 18]. It is very important to evaluate potential side effects via toxi￾cology tests in animals. Standard GLP non-clinical pharmacology, toxicity, and allergy studies for SBP and SBP-adjuvanted HBV vaccine were completed in rodents in the present study. The results demonstrate that SBP-adjuvanted HBV vaccine could enhance the production of anti-HBsAg antibody significantly. Throughout both acute (single dosage) and sub-acute (repeated dosage) studies, SBP and SBP-adjuvanted vaccine were well-tolerated. No treat￾ment-related effects in food consumption, body weight, clinical signs, body temperature, oph￾thalmic testing, or organ-body weight ratios were noted. White nodules were found at the injection site in the repeated-dosage vaccine group 3 days after the final injection. Histopathological examination of the nodules showed myofiber necro￾sis, mesenchyme inflammatory cell infiltration, macrophage aggregation, and abscesses. Major mechanical stimulation, including myofiber necrosis and mesenchyme inflammatory cell infil￾tration, was also observed in SBP group. Injection site reactions such as these are not uncom￾mon with adjuvanted vaccines: Lawrence Segal et al. reported that human papillomavirus vaccine could induce injection site reactions in rabbits and rats [18], and Penina Haber et al. showed that inactivated influenza vaccine induced injection site reactions in pre-licensure clinical trials [19, 20]. Some statistically significant changes in blood biochemistry parameters Fig 5. SBP adjuvanted hepatitis B vaccine could induce higher antibody titers and higher percentage of Th cells. Macaca fascicularis were immunized intramuscularly at days 1, 9, 15, 32 with vaccine alone (H) or together with SBP (H-S). (A) Blood samples were collected on indicated time points and HBsAg specific antibody titers were measured. (B)Percentage of CD4+ T cells was also measured at these time points. Results are expressed as the means ± SEM, * p<0.05. doi:10.1371/journal.pone.0170313.g005 A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 8 / 11