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 (HBsAgBinding 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 protein 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 vaccine. 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]. Specific 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 combine 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 effective innate and adaptive immune responses through increasing the ability of antigens to activate 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-inwater 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 reactions and increased IgE antibody responses [9]. Because of these limitations, there is an important 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 antibody 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 developing 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 laboratories. 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-planinternational-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 established 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 intravenously 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 continued 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 biochemistry 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. Fourteen days after the last injection, each guinea pig was injected intravenously with a double dosage 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 lymphocyte 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 performed 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 abnormalities 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 anatomy 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 SBPadjuvanted 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, common 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 SBPadjuvanted 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 SBPadjuvanted 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