@PLOS ONE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) Shanghai(13431900602,http://www.dragon-star.concernsaboutsideeffectsApprovedadjuvantsincludealuminumhydroxidetheoil-in au/callshangha-2013-st-innovation-action-plan: water emulsions MF59 16) and AS03 (Z), and alum with monophosphoryl lipid A(AS04)[8] international-cooperation-programme/ Although alum is considered a safe adjuvant in humans, it has been associated with local Competing Interests: The authors have declared tions and increased IgE antibody responses [2]. Because of these limitations, there is an that no competing interests exi tant need to identify novel adjuvants for HBv vaccines The novel HBsAg-binding protein, SBP, has been screened from a human liver cDNA pression library. Previous results show that, when combined with an HBV vaccine, SBPcan promote the uptake of HBsAg by antigen-presenting cells and enhance HBsAg-specific anti body production in BALB/c mice without any noticeable side effects [10]. These results suggest that SBP has the potential to be used as a novel adjuvant for HBv vaccine. We have been devel- oping a formulation of HBv vaccine consisting of HBsAg and SBP. The pharmacodynamics and safety of this new vaccine are evaluated normatively in the study presented here. Materials and methods Study design and vaccines We undertook a GLP pre-clinical allergic reaction test, acute and long term toxicity test [11, 12] to evaluate the safety of SBP and a candidate hepatitis B (Fig 1). SBP, HBsAg and new hepatitis B vaccine were produced by Dalian Hissen Bio-pharmaceuticals Company (Dalian, China). Each dose of SBP adjuvanted vaccine(H-S,05ml, Lot NO. 201501013S115 was a mixture of HBsAg(10ug), SBP(15ug)and aluminum(0. 22-0. 3 mg). The concentration of SBP solution(S, Lot NO. 20141212)was 30 ug/ml and each dose of general vaccine(H, 05ml, Lot NO. 201501013)was a mixture of HBsAg(10ug)and aluminum(0. 22-0.3 mg Animals and procedures Animal procurement and care. Sprague Dawley(SD)rats for pharmacodynamics studies were purchased from the Shanghai SLAC Laboratory Animal Company( Shanghai, China) nd maintained under specific pathogen free(SPF)conditions at Fudan University. For the good laboratory practice(GLP) safety evaluation, SD rats, Institute of Cancer Research(ICR) mice, and Hartley guinea pigs were purchased from Beijing Vital River Laboratory animal Technology(Beijing, China)and maintained under SPF conditions at Beijing JOINN laborato- ries. Macaca fascicularis for glp pharmacodynamics and safety studies were purchased from nd maintained under SPF conditions at the National Shanghai Center for Drug Safety CD(SD)Rat intramuscular injection 3o/Gr。up ICR mice intravenous bservation A Day 1 Day 15 Day 29 Day 43 Day 1 Day 28 Day o Day 15 intramuscular injection Post injection clinical observation Fig 1. Procedures of safety evaluation. (a) Experimental design of GLP non-clinical acute toxic test in ICR mice. b)Experimental design of GLP non-clinical long toxic test in rats. (c) Experimental design of GLP non-clinical allergic test in guinea pigs. doi: 10. 1371/journal pone. 0170313g001 PLOS ONE DO1: 10. 1371/journal pone. 0170313 January 19, 2017 2/concerns about side effects. Approved adjuvants include aluminum hydroxide, the oil-in￾water emulsions MF59 [6] and AS03 [7], and alum with monophosphoryl lipid A (AS04) [8]. Although alum is considered a safe adjuvant in humans, it has been associated with local reac￾tions and increased IgE antibody responses [9]. Because of these limitations, there is an impor￾tant need to identify novel adjuvants for HBV vaccines. The novel HBsAg-binding protein, SBP, has been screened from a human liver cDNA expression library. Previous results show that, when combined with an HBV vaccine, SBP can promote the uptake of HBsAg by antigen-presenting cells and enhance HBsAg-specific anti￾body production in BALB/c mice without any noticeable side effects [10]. These results suggest that SBP has the potential to be used as a novel adjuvant for HBV vaccine. We have been devel￾oping a formulation of HBV vaccine consisting of HBsAg and SBP. The pharmacodynamics and safety of this new vaccine are evaluated normatively in the study presented here. Materials and Methods Study design and vaccines We undertook a GLP pre-clinical allergic reaction test, acute and long term toxicity test [11, 12] to evaluate the safety of SBP and a candidate hepatitis B vaccine (Fig 1). SBP, HBsAg and new hepatitis B vaccine were produced by Dalian Hissen Bio-pharmaceuticals Company (Dalian, China). Each dose of SBP adjuvanted vaccine (H-S, 0.5ml, Lot NO. 201501013S115) was a mixture of HBsAg (10μg), SBP (15μg) and aluminum (0.22–0.3 mg). The concentration of SBP solution (S, Lot NO. 20141212) was 30 μg/ml and each dose of general vaccine (H, 0.5ml, Lot NO. 201501013) was a mixture of HBsAg (10μg) and aluminum (0.22–0.3 mg). Animals and procedures Animal procurement and care. Sprague Dawley (SD) rats for pharmacodynamics studies were purchased from the Shanghai SLAC Laboratory Animal Company (Shanghai, China) and maintained under specific pathogen free (SPF) conditions at Fudan University. For the good laboratory practice (GLP) safety evaluation, SD rats, Institute of Cancer Research (ICR) mice, and Hartley guinea pigs were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China) and maintained under SPF conditions at Beijing JOINN laborato￾ries. Macaca fascicularis for GLP pharmacodynamics and safety studies were purchased from and maintained under SPF conditions at the National Shanghai Center for Drug Safety Fig 1. Procedures of safety evaluation. (a) Experimental design of GLP non-clinical acute toxic test in ICR mice. (b) Experimental design of GLP non-clinical long toxic test in rats. (c) Experimental design of GLP non-clinical allergic test in guinea pigs. doi:10.1371/journal.pone.0170313.g001 A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 2 / 11 Shanghai (13431900602, http://www.dragon-star. eu/call/shanghai-2013-st-innovation-action-plan￾international-cooperation-programme/). Competing Interests: The authors have declared that no competing interests exist