圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) HBsAg group, anti-HBV IgG began to decline in the second month, while the antibody levels of the groups that received vaccine adjuvanted with SBP did not decline until the third mont SBP and vaccine did not induce acute toxicity in ICR mice animals. Institute of Cancer Research mice were used to evaluate the acute toxicity etector b Before clinical safety evaluation, vaccine development requires extensive pre-clinical testi SBP and SBP-adjuvanted HBV vaccine. No mice died during the study, and no clinical abnor malities in mental state, behavior, respiration, morbidity, secretions, feces, skin, eyes, ear, stomach, etc. were observed in the treated mice. There were also no significant differences between groups in body weight or food/water consumption(data not shown). The gross anat omy of each mouse was examined after they were sacrificed, and no abnormalities were observed in the main organs including brain, skin, liver, spleen, and kidneys SBP did not induce long-term toxicity in rats, but high dosages of SBP adjuvanted vaccine could cause local irritation For the long-term toxicity study, SD rats were injected repeatedly with SBP alone or SBP in sbp did not result in abnormal hanges in any of the tested parameters ( Table 1). For the groups that received vaccine, com mon irritation reactions, such as necrosis of the myofiber, were observed at the injection site As shown in Fig 3, lesions were still present at the end of the recovery period, indicating that the condition would likely take a long time to resolve Hematology results from the long-term toxicity test showed that female rats in the SBP d juvanted vaccine(0.5 mL) group had increased levels of neutrophils(cells important in innate immunity and anti-inflammation)3 days after the final injection. Male rats in the SBP of white blood cell that combats multicellular parasites and allergens), and monocytes(the ps adjuvanted vaccine(1.5 mL) group showed decreased levels of neutrophils, eosinophils(a tyE largest type of leukocyte, which can phagocytize foreign materials and play a role in immune response)(P< 0.05)(Tables A-D in SI File). The inflammatory changes at the injection sites (Fig 3)may be related the immunoreaction caused by the vaccine. Three days after the first injection, the level of reticulocytes(immature red blood cells which could reflect the red blood hematopoietic function of bone marrow) decreased in female rats for the SBP (0.5 mL) group, d the mean corpuscular hemoglobin concentration(often used to identify anemia) Table 1. Preliminary GLP pre-clinical long term toxicity tests in rats. ndex Administration period Recovery period Mortality I 1 death in H-S group(overdose anesthesia) None Clinical signs No difference No differe Normal Te ture Slightly change at 3 time points Slightly change at 1 time point No abnormalities No abnormalities Organ-body No abnormalities No abnormalities ratios Gross anatomy White nodule at injection part in H-S group nodule at injection part in H-s group jection part Necrosis of myofiber and mesenchyme inflammatory cell infiltration rophage aggregation and mesenchyme macrophage aggregation and abscess in H-s group matory cell infiltration in H-s group doi: 10. 1371/journal pone. 0170313.t001 PLOS ONE DO1: 10. 1371/journal pone. 0170313 January 19, 2017 5HBsAg group, anti-HBV IgG began to decline in the second month, while the antibody levels of the groups that received vaccine adjuvanted with SBP did not decline until the third month. SBP and vaccine did not induce acute toxicity in ICR mice Before clinical safety evaluation, vaccine development requires extensive pre-clinical testing in animals. Institute of Cancer Research mice were used to evaluate the acute toxicity effects of SBP and SBP-adjuvanted HBV vaccine. No mice died during the study, and no clinical abnor￾malities in mental state, behavior, respiration, morbidity, secretions, feces, skin, eyes, ear, stomach, etc. were observed in the treated mice. There were also no significant differences between groups in body weight or food/water consumption (data not shown). The gross anat￾omy of each mouse was examined after they were sacrificed, and no abnormalities were observed in the main organs including brain, skin, liver, spleen, and kidneys. SBP did not induce long-term toxicity in rats, but high dosages of SBP￾adjuvanted vaccine could cause local irritation For the long-term toxicity study, SD rats were injected repeatedly with SBP alone or SBP in combination with HBV vaccine. Generally, each dosage of SBP did not result in abnormal changes in any of the tested parameters (Table 1). For the groups that received vaccine, com￾mon irritation reactions, such as necrosis of the myofiber, were observed at the injection site. As shown in Fig 3, lesions were still present at the end of the recovery period, indicating that the condition would likely take a long time to resolve. Hematology results from the long-term toxicity test showed that female rats in the SBP￾adjuvanted vaccine (0.5 mL) group had increased levels of neutrophils (cells important in innate immunity and anti-inflammation) 3 days after the final injection. Male rats in the SBP￾adjuvanted vaccine (1.5 mL) group showed decreased levels of neutrophils, eosinophils (a type of white blood cell that combats multicellular parasites and allergens), and monocytes (the largest type of leukocyte, which can phagocytize foreign materials and play a role in immune response) (P < 0.05) (Tables A-D in S1 File). The inflammatory changes at the injection sites (Fig 3) may be related the immunoreaction caused by the vaccine. Three days after the first injection, the level of reticulocytes (immature red blood cells which could reflect the red blood hematopoietic function of bone marrow) decreased in female rats for the SBP (0.5 mL) group, and the mean corpuscular hemoglobin concentration (often used to identify anemia) Table 1. Preliminary GLP pre-clinical long term toxicity tests in rats. Index Administration period Recovery period Mortality 1 death in H-S group (overdose anesthesia) None Clinical signs No abnormalities No abnormalities Body weight No difference No difference Food consumption Normal Normal Temperature Slightly change at 3 time points Slightly change at 1 time point Ophthalmic testing No abnormalities No abnormalities Organ-body ratios No abnormalities No abnormalities Gross anatomy White nodule at injection part in H-S group White nodule at injection part in H-S group Injection part Necrosis of myofiber and mesenchyme inflammatory cell infiltration, macrophage aggregation and abscess in H-S group Macrophage aggregation and mesenchyme inflammatory cell infiltration in H-S group doi:10.1371/journal.pone.0170313.t001 A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 5 / 11