圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) Evaluation and Research. All procedures were performed according to the guidelines estab- lished by the National Institutes of Health, and every effort was made to minimize discomfort nd suffering on the part of the animals. The glp study was approved by the China Food and Drug Administration, and the other animal studies were approved by the Animal Experim Committee of Fudan University. All principles of laboratory animal care were followed Pharmacodynamics study. For the pharmacodynamics study, 60 SD rats(7-8 weeks of age)were randomly divided into 6 groups. Each rat received intramuscular injections 3 times at 2-week intervals. For each rat, the injections contained one of the following dosage: 1. 25 ug HBsAg, 2.5 ug HBsAg, 10 ug HBsAg, 1.25 ug HBsAg+ SBP, 2.5 ug HBsAg+ SBP, or 10 ug HBsAg+ SBP Serum samples were collected on days 0, 7, 14, 21, 28, 58, 88, and 118 following the first injection to observe the production of HBsAg-specific antibodies. Acute toxicity study. Eighty ICR mice(4-5 weeks of age)were divided into 4 groups Because the clinical administration of HB vaccine is intramuscularly injection, we choose nously injection of SBP group to evaluate the side effect of this new adjuvant. Each mouse intramuscularly injection as the major administration of drugs. Furthermore, we add intrave- received an injection of one of the following: normal saline, SBP(0.5 mL, intramuscularly injection), SBP(0.5 mL, intravenously injection)or HBsAg+SBP(0.5 mL, intramuscularly) To determine if any acute toxic reactions occurred, the animals were observed continuously for 4 h after the injection was administered. General and detailed clinical observation contin ued for 15 d thereafter. Then all animals were sacrificed and gross anatomy was performed to observe the lesion of different organs Long-term toxicity test. To study long-term toxic effects of the adjuvant, 150 SD rats (half males and half females, 7-8 weeks of age)were divided into 5 groups. Each rat was injected intramuscularly on days 0, 15, 29, and 43 with normal saline, SBP(0.5 mL or 1.5 mL), or HBsAg+ SBP (0.5 mL or 1.5 mL). Various parameters, including clinical signs, body weight, d temperature, were monitored to evaluate the possibility of long-term toxic reaction to SBP d vaccine. Blood samples were collected on days 3, 46, and 72 for hematology and biochem istry assays. Three days after the last injection, 20 rats(half males and half females)from each group were sacrificed for gross and histopathological examination. The remainder of the rats were sacrificed at the end of the recovery period for gross and histopathological examination Allergic reaction test. For the allergic reaction test, 54 male Hartley guinea pigs were divided into 6 groups. Guinea pigs were injected intramuscularly with normal saline(0.5 mL) human albumin(0.5 mL, 40 mg/mL), SBP (0.05 mL or 0.5 mL), or HBs Ag +SBP(0.05 or 0.5 mL)3 times at 2-day intervals in order to sensitize them to the corresponding antigens. Four- teen days after the last injection, each guinea pig was injected intravenously with a double dos age of the corresponding antigens and observed continuously for 30 min to record allergy Macaca fascicularis study. To evaluate the effects of SBP in Macaca fascicularis, animal were injected on days 1, 9, 15, and 32 with HBsAg(0.5 mL)or HBsAg+ SBP (0.5 mL). Blood samples were collected on days 0, 8, 14, 20, 31, and 37 for hematology, antibody titer, and lym- phocyte differentiation analysis Enzyme linked immunosorbent assay The HBs Ag specific serum antibody was measured using ELISA. Briefly, 100 ul/well diluted serum samples or rat antibody standards(Abcam, Cambridge, UK)were added into a 96-well plate, and following by incubation with HRP conjugated goat anti-mouse IgG antibodies (Santa Cruz, CA, USA). After washing, 100 ul/well TMB substrate(BD Biosciences, San PLOS ONE DO1: 10. 1371/journal pone. 0170313 JanuaryEvaluation and Research. All procedures were performed according to the guidelines estab￾lished by the National Institutes of Health, and every effort was made to minimize discomfort and suffering on the part of the animals. The GLP study was approved by the China Food and Drug Administration, and the other animal studies were approved by the Animal Experiment Committee of Fudan University. All principles of laboratory animal care were followed precisely. Pharmacodynamics study. For the pharmacodynamics study, 60 SD rats (7–8 weeks of age) were randomly divided into 6 groups. Each rat received intramuscular injections 3 times at 2-week intervals. For each rat, the injections contained one of the following dosage: 1.25 μg HBsAg, 2.5 μg HBsAg, 10 μg HBsAg, 1.25 μg HBsAg + SBP, 2.5 μg HBsAg + SBP, or 10 μg HBsAg + SBP. Serum samples were collected on days 0, 7, 14, 21, 28, 58, 88, and 118 following the first injection to observe the production of HBsAg-specific antibodies. Acute toxicity study. Eighty ICR mice (4–5 weeks of age) were divided into 4 groups. Because the clinical administration of HB vaccine is intramuscularly injection, we choose intramuscularly injection as the major administration of drugs. Furthermore, we add intrave￾nously injection of SBP group to evaluate the side effect of this new adjuvant. Each mouse received an injection of one of the following: normal saline, SBP (0.5 mL, intramuscularly injection), SBP (0.5 mL, intravenously injection) or HBsAg + SBP (0.5 mL, intramuscularly). To determine if any acute toxic reactions occurred, the animals were observed continuously for 4 h after the injection was administered. General and detailed clinical observation contin￾ued for 15 d thereafter. Then all animals were sacrificed and gross anatomy was performed to observe the lesion of different organs. Long-term toxicity test. To study long-term toxic effects of the adjuvant, 150 SD rats (half males and half females, 7–8 weeks of age) were divided into 5 groups. Each rat was injected intramuscularly on days 0, 15, 29, and 43 with normal saline, SBP (0.5 mL or 1.5 mL), or HBsAg + SBP (0.5 mL or 1.5 mL). Various parameters, including clinical signs, body weight, and temperature, were monitored to evaluate the possibility of long-term toxic reaction to SBP and vaccine. Blood samples were collected on days 3, 46, and 72 for hematology and biochem￾istry assays. Three days after the last injection, 20 rats (half males and half females) from each group were sacrificed for gross and histopathological examination. The remainder of the rats were sacrificed at the end of the recovery period for gross and histopathological examination. Allergic reaction test. For the allergic reaction test, 54 male Hartley guinea pigs were divided into 6 groups. Guinea pigs were injected intramuscularly with normal saline (0.5 mL), human albumin (0.5 mL, 40 mg/mL), SBP (0.05 mL or 0.5 mL), or HBsAg + SBP (0.05 or 0.5 mL) 3 times at 2-day intervals in order to sensitize them to the corresponding antigens. Four￾teen days after the last injection, each guinea pig was injected intravenously with a double dos￾age of the corresponding antigens and observed continuously for 30 min to record allergy symptoms. Macaca fascicularisstudy. To evaluate the effects of SBP in Macaca fascicularis, animals were injected on days 1, 9, 15, and 32 with HBsAg (0.5 mL) or HBsAg + SBP (0.5 mL). Blood samples were collected on days 0, 8, 14, 20, 31, and 37 for hematology, antibody titer, and lym￾phocyte differentiation analysis. Enzyme linked immunosorbent assay The HBsAg specific serum antibody was measured using ELISA. Briefly, 100 μl/well diluted serum samples or rat antibody standards (Abcam, Cambridge, UK) were added into a 96-well plate, and following by incubation with HRP conjugated goat anti-mouse IgG antibodies (Santa Cruz, CA, USA). After washing, 100 μl/well TMB substrate (BD Biosciences, San Diego, A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 3 / 11