圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) were observed in the vaccine group 3 days after the final injection, which we considered to be associated with the inflammatory response at the injection site. All other differences were con- sidered incidental and within the normal reference ranges for the animals, and therefore of no toxicological significan Allergic sensitization of drugs is an important health problem with serious clinical conse [21, 22]. In the pre-clinical evaluation process, guinea pig models are often used to evaluate the allergenicity of drugs [23, 24]. Our results show that SBP could induce strong sys temic active allergic reaction under the conditions used in this study. Considering that SBP has been shown to be an immunogenic protein for guinea pigs at a sensitization dosage that is s0 (low dosage)and 150(high dosage) times the clinical dosage these results may not indicate a serious allergy concern for humans. To further evaluate the effects of SBP, we used Macaca fascicularis as a model animal because of its close genetic relationship to humans [25]. Using an immunizing dosage that was 50 times the clinical dosage, no abnormalities of toxicological significance were found. Our esults showed that SBP could trigger antibody responses as a result of co-immunization with HBV vaccine. Moreover, SBP-adjuvanted vaccine induced an increase in the number of CD4+ T cells, which may correlate with antibody production [26] The importance of developing safer and better vaccine adjuvants for human use is indisput- able, but it is difficult to gain approval for adjuvants as a final product. This study comprehen sively evaluated the adjuvanticity and safety of SBP in regard to HBV vaccine. The results demonstrate that SBP could enhance the humoral response elicited by the existing vaccine Given the encouraging safety data obtained in this study, further evaluation of SBP as a vaccine adjuvant for human use is warranted. This research has the potential to accelerate adjuvant development for HBV vaccine and for other vaccine types in the future Supporting Informa SI File. Tables A-D; Hematology and clinical chemistry parameter changes in rats injected with different agents after 46 days. Values are expressed as meantstandard deviation ' indi cates significant increase or decrease in p values (DOCX) Acknowledgments This work was supported by the National Major Scientific and Technological Special Project (2012ZX10002006-002-003), the National High Technology Research and Development Pro gram of China(2011AA02A114), the National Natural Science Foundation of China (31370927 and 30571650)and the Science and Technology Innovation Action Plan of Shang hai(13431900602) Author contributions Funding acqu Investigation: JW. Methodology: NZ Cs PLOS ONE DO1: 10. 1371/journal pone. 0170313 January 19, 2017were observed in the vaccine group 3 days after the final injection, which we considered to be associated with the inflammatory response at the injection site. All other differences were con￾sidered incidental and within the normal reference ranges for the animals, and therefore of no toxicological significance. Allergic sensitization of drugs is an important health problem with serious clinical conse￾quences [21, 22]. In the pre-clinical evaluation process, guinea pig models are often used to evaluate the allergenicity of drugs [23, 24]. Our results show that SBP could induce strong sys￾temic active allergic reaction under the conditions used in this study. Considering that SBP has been shown to be an immunogenic protein for guinea pigs at a sensitization dosage that is 50 (low dosage) and 150 (high dosage) times the clinical dosage these results may not indicate a serious allergy concern for humans. To further evaluate the effects of SBP, we used Macaca fascicularis as a model animal because of its close genetic relationship to humans [25]. Using an immunizing dosage that was 50 times the clinical dosage, no abnormalities of toxicological significance were found. Our results showed that SBP could trigger antibody responses as a result of co-immunization with HBV vaccine. Moreover, SBP-adjuvanted vaccine induced an increase in the number of CD4+ T cells, which may correlate with antibody production [26]. The importance of developing safer and better vaccine adjuvants for human use is indisput￾able, but it is difficult to gain approval for adjuvants as a final product. This study comprehen￾sively evaluated the adjuvanticity and safety of SBP in regard to HBV vaccine. The results demonstrate that SBP could enhance the humoral response elicited by the existing vaccine. Given the encouraging safety data obtained in this study, further evaluation of SBP as a vaccine adjuvant for human use is warranted. This research has the potential to accelerate adjuvant development for HBV vaccine and for other vaccine types in the future. Supporting Information S1 File. Tables A-D; Hematology and clinical chemistry parameter changes in rats injected with different agents after 46 days. Values are expressed as mean±standard deviation. indi￾cates significant increase or decrease in p values. (DOCX) Acknowledgments This work was supported by the National Major Scientific and Technological Special Project (2012ZX10002006-002-003), the National High Technology Research and Development Pro￾gram of China (2011AA02A114), the National Natural Science Foundation of China (31370927 and 30571650) and the Science and Technology Innovation Action Plan of Shang￾hai (13431900602). Author Contributions Conceptualization: NZ. Data curation: JW. Formal analysis: JW. Funding acquisition: NZ. Investigation: JW. Methodology: NZ CS. A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 9 / 11