复旦大学：《免疫学 Immunology》教学论文_A Pre-Clinical Safety Evaluation of SBP（HBsAg-BindingProtein）Adjuvant for Hepatitis BVaccine

圆PLoS|oNE RESEARCH ARTICLE A Pre-Clinical Safety Evaluation of SBP(HBSAg Binding Protein) Adjuvant for Hepatitis B Vaccine Jingbo Wang, Caixia Su, Rui Liu, Baoxiu Liu, Inam Ullah Khan, Jun Xie*, Naishuo Zhu Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, Institute of Biomedical Science, School of Life Sciences, Fudan University, Shanghai, China zhu@fudan.edu.cn(NZ);xiejun@fudan.edu.cn(JX) Check for Abstract Although adjuvants are a common component of many vaccines, there are few adjuvants licensed for use in humans due to concerns about their toxic effects There is a need to develop new and safe adjuvants, because some existing vaccines have low immunogenicity OpenACCESS among certain patient groups. In this study, SBP, a hepatitis B surface antigen binding pro- tein that was discovered through screening a human liver cDNA expression library, was Citation: Wang J, Su C, Liu R, Liu B, Khan IU, Xie introduced into hepatitis B vaccine. a good laboratory practice, non-clinical safety evaluation SBP (HBsAg-Binding Protein) Adjuvant for was performed to identify the side effects of both SBP and SBP-adjuvanted hepatitis B vac- PLOS ONE 12(1): 0170313. cine. The results indicate that SBP could enhance the HBsAg-specific thus increasing the protection provided by the hepatitis B vaccine. The safety data obtained Editor: Isabelle A Chemin, Centre de Recherche en here warrant further investigation of SBP as a vaccine adjuvant Cancerologie de lyon, FRANCE Accepted: January 2, 2017 Published: January 19, 2017 Introduction Copyright: 2017 Wang et a. This is an open The infectious disease hepatitis B, which is caused by the hepatitis B virus(HBv), has troubled Creative Commons Attribution License, which people worldwide for many years. The current prophylactic HBV vaccines, which are based on permits unrestricted use, distribution, and recombinant hepatitis B surface antigen(HBsAg), have successfully decreased rates of HB reproduction in any medium, provided the original infection and transmission. However, there are more than 2 billion people who have been author and source are credited infected with HBV and are therefore at high risk for liver failure, cirrhosis, or cancer [1]. Spe- Data Availability Statement: All relevant data are cific groups of patients respond poorly or not at all to conventional HBv vaccines. Because of within the paper. the poor immunogenicity of HBsAg, new methods are needed to improve the ability of the Funding: This work was supported by the National HBV vaccine to trigger protective immunity [2, 3]. Third-generation HBV vaccines that com- Major Scientific and Technological Special Project bine small S antigen with PreSl and PreS2 antigens have been shown to induce a stronger (2012zx10002006-002-003,http://www.nmp.ov.immuneresponseinnon-andlowrespondersthancurrentHbvvaccines[4].Conventionally xis/zdy/201012/20101208 2128 htm), the used adjuvants, such as aluminum salts, allow for persistent release of the antigen, delaying ational High Technology Research and clearance and resulting in more exposure to the immune system [5]. Adjuvants can elicit effec- http:/program.mostgov.cn),theNationalNaturaltiveinnateandadaptiveimmuneresponsesthroughincreasingtheabilityofantigenstoacti cience Foundation of China(31370927 vate signaling pathways 30571650,http://www.nsfc.gov.cndandthe Although adjuvants improve vaccine formulations and lead to better and more controllable Science and Technology Innovation Action Plan of immune responses, very few adjuvants have been licensed for use in humans because of PLOS ONE DO1: 10. 1371/journal pone. 0170313 January

RESEARCH ARTICLE A Pre-Clinical Safety Evaluation of SBP (HBsAg￾Binding Protein) Adjuvant for Hepatitis B Vaccine Jingbo Wang, Caixia Su, Rui Liu, Baoxiu Liu, Inam Ullah Khan, Jun Xie*, Naishuo Zhu* Laboratory of Molecular Immunology, State Key Laboratory of Genetic Engineering, Institute of Biomedical Science, School of Life Sciences, Fudan University, Shanghai, China * nzhu@fudan.edu.cn (NZ); xiejun@fudan.edu.cn (JX) Abstract Although adjuvants are a common component of many vaccines, there are few adjuvants licensed for use in humans due to concerns about their toxic effects. There is a need to develop new and safe adjuvants, because some existing vaccines have low immunogenicity among certain patient groups. In this study, SBP, a hepatitis B surface antigen binding pro￾tein that was discovered through screening a human liver cDNA expression library, was introduced into hepatitis B vaccine. A good laboratory practice, non-clinical safety evaluation was performed to identify the side effects of both SBP and SBP-adjuvanted hepatitis B vac￾cine. The results indicate that SBP could enhance the HBsAg-specific immune response, thus increasing the protection provided by the hepatitis B vaccine. The safety data obtained here warrant further investigation of SBP as a vaccine adjuvant. Introduction The infectious disease hepatitis B, which is caused by the hepatitis B virus (HBV), has troubled people worldwide for many years. The current prophylactic HBV vaccines, which are based on recombinant hepatitis B surface antigen (HBsAg), have successfully decreased rates of HBV infection and transmission. However, there are more than 2 billion people who have been infected with HBV and are therefore at high risk for liver failure, cirrhosis, or cancer [1]. Spe￾cific groups of patients respond poorly or not at all to conventional HBV vaccines. Because of the poor immunogenicity of HBsAg, new methods are needed to improve the ability of the HBV vaccine to trigger protective immunity [2, 3]. Third-generation HBV vaccines that com￾bine small S antigen with PreS1 and PreS2 antigens have been shown to induce a stronger immune response in non- and low responders than current HBV vaccines [4]. Conventionally used adjuvants, such as aluminum salts, allow for persistent release of the antigen, delaying clearance and resulting in more exposure to the immune system [5]. Adjuvants can elicit effec￾tive innate and adaptive immune responses through increasing the ability of antigens to acti￾vate signaling pathways. Although adjuvants improve vaccine formulations and lead to better and more controllable immune responses, very few adjuvants have been licensed for use in humans because of PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 1 / 11 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Wang J, Su C, Liu R, Liu B, Khan IU, Xie J, et al. (2017) A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) Adjuvant for Hepatitis B Vaccine. PLoS ONE 12(1): e0170313. doi:10.1371/journal.pone.0170313 Editor: Isabelle A Chemin, Centre de Recherche en Cancerologie de Lyon, FRANCE Received: July 27, 2016 Accepted: January 2, 2017 Published: January 19, 2017 Copyright: © 2017 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the National Major Scientific and Technological Special Project (2012ZX10002006-002-003, http://www.nmp.gov. cn/zxjs/zdxy/201012/t20101208_2128.htm), the National High Technology Research and Development Program of China (2011AA02A114, http://program.most.gov.cn/), the National Natural Science Foundation of China (31370927 and 30571650, http://www.nsfc.gov.cn/) and the Science and Technology Innovation Action Plan of

@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

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 January

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 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

圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) CA, USA)was added and the reactions were terminated by the addition of 50 ul/well 2M H2S04. The absorbance at 450nm was immediately read on a microplate reader(Molecular devices, Sunnyvale, CA, USA). A standard curve of IgG standard was used to transform the absorbance to antibody concentration Flow cytometry For lymphocytes differentiation, blood samples were harvested at indicated time points and monocytes were separated though isodensity centrifugation. Thereafter, approximately 10 lls were re suspended in 100 uL of PBS and incubated with 2 ug anti-CD3c FITC together with anti-CD4 PE or anti-CD8 PE antibodies(all from BioLegend, San Diego, CA, USA)for 20 min on ice. Thereafter. the cells were washed twice with pbs and fle formed using a FACSort instrument(BD Biosciences, San Jose, CA, USA). Data was analyze ng FlowJo software(Tree Star, San Caros, CA, USA) Statistical analysis The statistical significance of the results was determined using the Provantis system(SAS, US) The homogeneity of variance was examined with Levene's test A one-way aNOVa was used to determine the significance of the data(P>0.05). For groups in which P <0.05, Dunnetts test was used for comparative analysis. For groups with heterogeneity of variance, Levene's test was used after log transforming the data. The Kruskal-Wallis test was used for data that still had heterogeneity of variance after log transformation. When evaluating differences between experimental and control samples, P<0.05 was considered to be statistically significant. Results SBP adjuvant enhances HBsAg-specific antibody production in rats The serum antibody titer correlates with the protection offered by vaccines. To determine the ffects of SBP on the immunogenicity of HBV vaccine, HBs Ag-specific antibody was assessed by enzyme-linked immunosorbent assay in rats that received various dosages of HBs Ag with or without the SBP adjuvant. Higher dosages of HBs Ag adjuvanted with SBP triggered more Igg generation in rats after day 14, and IgG production doubled on day 21 compared with the non-adjuvanted group(Fig 2). For the lowest dosage group, an increase in IgG was first detected on day 21, and the rats that received SBP had higher levels of IgG production. The results also show that SBP plays a role in the maintenance of antibody levels over time. In the HBsAg dose: 10ug HBsAg dose: 2. 5ug HBsAg dose: 1. 25ug Fig 2. HBsAg specific antibody induced by sBP adjuvanted and non-adjuvanted es. rats were collected on indicated time points and anti-HBs total lgG was measured by ELISA Results ale.Nether were immunized intramuscularly 3 times at a 2-week interval with vaccine alone(H)or tog with SBP(H-S). There are 6 groups with 3 injected HBsAg dose: 1.25ug, 2.5ug and 10ug. Blood sar doi: 10.1371/journal pone. 0170313. g002 PLOS ONE DO1: 10. 1371/journal pone. 0170313 January 4/1

CA, USA) was added and the reactions were terminated by the addition of 50 μl/well 2M H2SO4. The absorbance at 450nm was immediately read on a microplate reader (Molecular devices, Sunnyvale, CA, USA). A standard curve of IgG standard was used to transform the absorbance to antibody concentration. Flow cytometry For lymphocytes differentiation, blood samples were harvested at indicated time points and monocytes were separated though isodensity centrifugation. Thereafter, approximately 106 cells were re suspended in 100 μL of PBS and incubated with 2 μg anti-CD3c FITC together with anti-CD4 PE or anti-CD8 PE antibodies (all from BioLegend, San Diego, CA, USA) for 20 min on ice. Thereafter, the cells were washed twice with PBS and flow cytometry was per￾formed using a FACSort instrument (BD Biosciences, San Jose, CA, USA). Data was analyzed using FlowJo software (Tree Star, San Caros, CA, USA). Statistical analysis The statistical significance of the results was determined using the Provantis system (SAS, US). The homogeneity of variance was examined with Levene’s test. A one-way ANOVA was used to determine the significance of the data (P > 0.05). For groups in which P  0.05, Dunnett’s test was used for comparative analysis. For groups with heterogeneity of variance, Levene’s test was used after log transforming the data. The Kruskal-Wallis test was used for data that still had heterogeneity of variance after log transformation. When evaluating differences between experimental and control samples, P  0.05 was considered to be statistically significant. Results SBP adjuvant enhances HBsAg-specific antibody production in rats The serum antibody titer correlates with the protection offered by vaccines. To determine the effects of SBP on the immunogenicity of HBV vaccine, HBsAg-specific antibody was assessed by enzyme-linked immunosorbent assay in rats that received various dosages of HBsAg with or without the SBP adjuvant. Higher dosages of HBsAg adjuvanted with SBP triggered more IgG generation in rats after day 14, and IgG production doubled on day 21 compared with the non-adjuvanted group (Fig 2). For the lowest dosage group, an increase in IgG was first detected on day 21, and the rats that received SBP had higher levels of IgG production. The results also show that SBP plays a role in the maintenance of antibody levels over time. In the Fig 2. HBsAg specific antibody induced by SBP adjuvanted and non-adjuvanted vaccines. Rats (n = 10/group) were immunized intramuscularly 3 times at a 2-week interval with vaccine alone (H) or together with SBP (H-S). There are 6 groups with 3 injected HBsAg dose: 1.25μg, 2.5μg and 10μg. Blood samples were collected on indicated time points and anti-HBs total IgG was measured by ELISA. Results are expressed as the means ± SEM, * p<0.05. doi:10.1371/journal.pone.0170313.g002 A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 4 / 11

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 5

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 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

圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) a x 200(HE staining).(a)Ns group, 3 days after the last injection, normal tissue. (b)V+SBP(high dose)group, 3 days after t necrosis of myofiber(focal). (c)V+SBP (high dose)group, 3 days after the last injection, mesenchyme inflammatory cell int macrophage aggregation(multi-focus).(d)V+SBP(high dose)group, at the end of the recovery period, mesenchyme inflammatory cell infiltration and macrophage aggregation(multi-focus) doi: 10. 1371/journal pone. 0170313.g003 decreased in male rats from the SBP(1.5 mL)and SBP-adjuvanted vaccine(0.5 mL) groups ( data not shown). Although these changes were statistically significant, there was no clear cor relation with injection dosage or time. It is likely that these data do not have toxicological SBP and high-dosage SBP-adjuvanted vaccine could cause allergic reaction in guinea pigs The allergenicity of SBP and SBP-adjuvanted vaccine was evaluated in guinea pigs. No abnor malities were observed during the sensitization period. After the antigens were administered, different levels of active systemic allergic reaction were observed in both SBP groups. Further- more, the higher dosage of SBP-adjuvanted vaccine also caused active systemic allergic reac- tions ( table 2 SBP could induce the production of SBP-specific antibodies in rats As a foreign protein for rats, SBP could induce antibody production after injection. In both SBP groups, SBP-specific antibodies were detected on day 21, after 2 injections, and declined 7 PLOS ONE DO1: 10. 1371/journal pone. 0170313 January

decreased in male rats from the SBP (1.5 mL) and SBP-adjuvanted vaccine (0.5 mL) groups (data not shown). Although these changes were statistically significant, there was no clear cor￾relation with injection dosage or time. It is likely that these data do not have toxicological meaning. SBP and high-dosage SBP-adjuvanted vaccine could cause allergic reaction in guinea pigs The allergenicity of SBP and SBP-adjuvanted vaccine was evaluated in guinea pigs. No abnor￾malities were observed during the sensitization period. After the antigens were administered, different levels of active systemic allergic reaction were observed in both SBP groups. Further￾more, the higher dosage of SBP-adjuvanted vaccine also caused active systemic allergic reac￾tions (Table 2). SBP could induce the production of SBP-specific antibodies in rats As a foreign protein for rats, SBP could induce antibody production after injection. In both SBP groups, SBP-specific antibodies were detected on day 21, after 2 injections, and declined 7 Fig 3. SBP could induce local irritation reaction. Representative photomicrographs of treated site are shown with original magnification: × 200 (HE staining). (a) NS group, 3 days after the last injection, normal tissue. (b) V+SBP (high dose) group, 3 days after the last injection, necrosis of myofiber (focal). (c) V+SBP (high dose) group, 3 days after the last injection, mesenchyme inflammatory cell infiltration and macrophage aggregation (multi-focus). (d) V+SBP (high dose) group, at the end of the recovery period, mesenchyme inflammatory cell infiltration and macrophage aggregation (multi-focus). doi:10.1371/journal.pone.0170313.g003 A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 6 / 11

圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) Table 2. GLP allergic reaction test in guinea pig Group Positive rate Extent of reaction Negative control Positive control ~十十十 s(05m) 044 Hs(0.5m) ~十十十 negative allergy: + weakly positive allergy, ++ positive allergy, +++ strong positive allergy, ++++ do:10.1371/ ournal pone017031302 days afterwards(Fig 4). For SBP-adjuvanted vaccine groups, the lower dosage appeared to induce higher antibody production according to our results. It seems that the SBP-specific ntibody was produced, and then levels declined in a dosage - independent manner. The detailed mechanism is not yet clear. SBP-adjuvanted HBV vaccine could induce stronger immune response in Macaca fascicularis than unadjuvanted vaccine, without any side Macaca fascicularis were used to determine the effects of SBP-adjuvanted vaccine in primates As shown in Fig 5, animals that received SBP-adjuvanted vaccine had higher antibody levels after day 30 than the animals that received non-adjuvanted vaccine. This suggests that SBP nt to the hpv 1.5 S(0.5m) S(1.5m) 1.0 Hs(0.5m) HS(1.5m) 0.5 0.0 0d7d14d21d28d42d72d cific antibody production in rats. Rats (n= 10)were immunized intramuscularly 3 times at a 2-week interval with vacci ether with SBP (H-S). Blood samples were collected on indicated time points and anti-SBP total lgG was measured by 101371/ ournal pone.0170313 PLOS ONE DO1: 10. 1371/journal pone. 0170313 January 19, 2017

days afterwards (Fig 4). For SBP-adjuvanted vaccine groups, the lower dosage appeared to induce higher antibody production according to our results. It seems that the SBP-specific antibody was produced, and then levels declined in a dosage-independent manner. The detailed mechanism is not yet clear. SBP-adjuvanted HBV vaccine could induce stronger immune response in Macaca fascicularis than unadjuvanted vaccine, without any side effects Macaca fascicularis were used to determine the effects of SBP-adjuvanted vaccine in primates. As shown in Fig 5, animals that received SBP-adjuvanted vaccine had higher antibody levels after day 30 than the animals that received non-adjuvanted vaccine. This suggests that SBP could be a good adjuvant to the HPV vaccine. Table 2. GLP allergic reaction test in guinea pig. Group Positive rate Extent of reaction Negative control 0 - Positive control 1 +~+++ S (0.5ml) 0.44 +~++ S (1.5ml) 1 +~++++ H-S (0.5ml) 0 - H-S (1.5ml) 0.67 +~+++ –, negative allergy; +, weakly positive allergy; ++, positive allergy; +++, strong positive allergy; ++++, extremely positive allergy. doi:10.1371/journal.pone.0170313.t002 Fig 4. SBP-specific antibody production in rats. Rats (n = 10) were immunized intramuscularly 3 times at a 2-week interval with vaccine alone (H) or together with SBP (H-S). Blood samples were collected on indicated time points and anti-SBP total IgG was measured by ELISA. Results are expressed as the means ± SEM. * p<0.05. doi:10.1371/journal.pone.0170313.g004 A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 7 / 11

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, 2017

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 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

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, 2017

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 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

圆PLoS|oNE A Pre-Clinical Safety Evaluation of SBP(HBsAg-Binding Protein) Project administration: NZ JX. Resources: CS BL Validation: BL JW Writing-original draft: JW Writing- review editing: JW IK. References 1. Wei MJ, Pan XN, Wei KP, Li XH, Liu XL, Zhang XM, et al. Efficacy of HBV-pulsed DCs in combination with entecavir in patients with chronic hepatitis B infection. International immunopharmacology 2015: 27(2)23843.do:10.1016/ . intim201506019PMD:26118632 2. Hall S, Desbrow B, Anoopkumar-Dukie S, Davey AK, Arora D, McDermott C, et al. A review of the bio- activity of coffee, caffeine and key coffee constituents on inflammatory responses linked to depression Food Research International. 2015: 76: 626-36. 3. Janssen JM, Jackson S, Heyward WL, Janssen RS Immunogenicity of an investigational hepatitis B vaccine with a toll-like receptor 9 agonist adjuvant(HBsAg-1018)compared with a licensed hepatitis B vaccine in subpopulations of healthy adults 18-70 years of age. Vaccine. 2015: 33(31): 3614-8. doi: 10 1016/ vaccine201505070PMD:26067185 duction of a robust T- n-andlow-responders to conventional vaccination against hepatitis B byusir third generation PreS/S vaccine. Vaccine. 2014: 32: 5077-5082. doi: 10.1016/ vaccine. 2014.06.076 PMID:24975813 5. LiX, Thakkar SG, Ruwona TB, Williams RO 3rd, Cui Z. A method of lyophilizing vaccines containing aluminum salts into a dry powder without causing particle aggregation or decreasing the immunogenic ity following reconstitution Joumal of controlled release: official journal of the Controlled Release Soci- ety.2015;204385 6. Nakaya HI, Clutterbuck E, Kazmin D, Wang L, Cortese M, Bosinger SE, et aL. Systems biology of immu Proceedings of the National Academy of Sciences of the United States of America. 2016: 113(7): 1853- 8.doi:101073pnas1519690113PMD:26755593 7. Yam KK, Gupta J Allen EK, Burt KR, Beaulieu E, Mallett CP, et al. Comparison of As03 and Alum n immune responses elicited by AH3N2 split influenza vaccine in young, mature and aged BALB/c mi Vaccine.2016:34(12):14451.doi:10.1016/ vaccine201602012PMD:26873056 8. Didierlaurent AM, Morel S, Lockman L, Giannini SL, Bisteau M, carlsen H, et al. aso4, an aluminum salt-and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity Journal of immunology. 2009: 183(10): 6186-97. journa.2015:8(1):7.doi:10.1186/s4041301500605PMD:25780491 10. Zhang C, Gong L, Chen Y, Wang X, Zhu S, LiD, et al. A human-derived protein SBP(HBsAg- binding protein)can bind to hepatitis B virus surface antigen(HBsAg) and enhance the immune response to vaccine. Molecular Immunology 2013: 53 (1-2): 60-71. doi: 10. 1016/. molimm. 201206014PMID:22809814 11. Segal L, Wouters S, Morelle D, Gautier G, Le Gal J, Martin T, et al. Non-clinical safety and biodistribu- on of As03-adjuvanted inactivated pandemic influenza vaccines. Joumal of applied toxicology: JAT. 2015:35(12:1564-76.doi:10.1002/at3130PMD:25727696 nary safety an Y, TsukuiT, Ishii KJ, Coban C Current status of synthetic hemozoin adjuvant: A prelimi- ty evaluation Vaccine 2016: 34(18): 2055-61. doi: 10. 1016/ vaccine. 2016.02.064 PMID 13.:860Paem PLOS ONE DO1: 10. 1371/journal pone. 0170313 January

Project administration: NZ JX. Resources: CS BL. Software: JW. Supervision: NZ. Validation: BL JW. Visualization: JW. Writing – original draft: JW. Writing – review & editing: JW IK. References 1. Wei MJ, Pan XN, Wei KP, Li XH, Liu XL, Zhang XM, et al. Efficacy of HBV-pulsed DCs in combination with entecavir in patients with chronic hepatitis B infection. International immunopharmacology. 2015; 27(2):238–43. doi: 10.1016/j.intimp.2015.06.019 PMID: 26118632 2. Hall S, Desbrow B, Anoopkumar-Dukie S, Davey AK, Arora D, McDermott C, et al. A review of the bio￾activity of coffee, caffeine and key coffee constituents on inflammatory responses linked to depression. Food Research International. 2015; 76:626–36. 3. Janssen JM, Jackson S, Heyward WL, Janssen RS. Immunogenicity of an investigational hepatitis B vaccine with a toll-like receptor 9 agonist adjuvant (HBsAg-1018) compared with a licensed hepatitis B vaccine in subpopulations of healthy adults 18–70 years of age. Vaccine. 2015; 33(31):3614–8. doi: 10. 1016/j.vaccine.2015.05.070 PMID: 26067185 4. Krawczyk Adalbert, Ludwig Charlotte, Jochum Christoph et al. Induction of a robust T- and B-cell immune response in non- andlow-responders to conventional vaccination against hepatitis B byusing a third generation PreS/S vaccine. Vaccine. 2014; 32:5077–5082. doi: 10.1016/j.vaccine.2014.06.076 PMID: 24975813 5. Li X, Thakkar SG, Ruwona TB, Williams RO 3rd, Cui Z. A method of lyophilizing vaccines containing aluminum salts into a dry powder without causing particle aggregation or decreasing the immunogenic￾ity following reconstitution. Journal of controlled release: official journal of the Controlled Release Soci￾ety. 2015; 204:38–50. 6. Nakaya HI, Clutterbuck E, Kazmin D, Wang L, Cortese M, Bosinger SE, et al. Systems biology of immu￾nity to MF59-adjuvanted versus nonadjuvanted trivalent seasonal influenza vaccines in early childhood. Proceedings of the National Academy of Sciences of the United States of America. 2016; 113(7):1853– 8. doi: 10.1073/pnas.1519690113 PMID: 26755593 7. Yam KK, Gupta J, Allen EK, Burt KR, Beaulieu E, Mallett CP, et al. Comparison of AS03 and Alum on immune responses elicited by A/H3N2 split influenza vaccine in young, mature and aged BALB/c mice. Vaccine. 2016; 34(12):1444–51. doi: 10.1016/j.vaccine.2016.02.012 PMID: 26873056 8. Didierlaurent AM, Morel S, Lockman L, Giannini SL, Bisteau M, Carlsen H, et al. AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity. Journal of immunology. 2009; 183(10):6186–97. 9. Jensen-Jarolim E. Aluminium in Allergies and Allergen immunotherapy. The World Allergy Organization journal. 2015; 8(1):7. doi: 10.1186/s40413-015-0060-5 PMID: 25780491 10. Zhang C, Gong L, Chen Y, Wang X, Zhu S, Li D, et al. A human-derived protein SBP (HBsAg-binding protein) can bind to hepatitis B virus surface antigen (HBsAg) and enhance the immune response to hepatitis B virus (HBV) vaccine. Molecular Immunology. 2013; 53(1–2):60–71. doi: 10.1016/j.molimm. 2012.06.014 PMID: 22809814 11. Segal L, Wouters S, Morelle D, Gautier G, Le Gal J, Martin T, et al. Non-clinical safety and biodistribu￾tion of AS03-adjuvanted inactivated pandemic influenza vaccines. Journal of applied toxicology: JAT. 2015; 35(12):1564–76. doi: 10.1002/jat.3130 PMID: 25727696 12. Lee MS, Igari Y, Tsukui T, Ishii KJ, Coban C. Current status of synthetic hemozoin adjuvant: A prelimi￾nary safety evaluation. Vaccine. 2016; 34(18):2055–61. doi: 10.1016/j.vaccine.2016.02.064 PMID: 26976665 13. Block TM, Rawat S, Brosgart CL. Chronic hepatitis B: A wave of new therapies on the horizon. Antiviral research. 2015; 121:69–81. doi: 10.1016/j.antiviral.2015.06.014 PMID: 26112647 A Pre-Clinical Safety Evaluation of SBP (HBsAg-Binding Protein) PLOS ONE | DOI:10.1371/journal.pone.0170313 January 19, 2017 10 / 11