Article(id=1208518760273678561, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208518757253779608, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2021.12.02, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1616947200000, receivedDateStr=2021-03-29, revisedDate=1628438400000, revisedDateStr=2021-08-09, acceptedDate=null, acceptedDateStr=null, onlineDate=1766062925642, onlineDateStr=2025-12-18, pubDate=1640620800000, pubDateStr=2021-12-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1766062925642, onlineIssueDateStr=2025-12-18, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1766062925642, creator=13701087609, updateTime=1766062925642, updator=13701087609, issue=Issue{id=1208518757253779608, tenantId=1146029695717560320, journalId=1189873630562394117, year='2021', volume='46', issue='12', pageStart='1167', pageEnd='1267', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1766062924920, creator=13701087609, updateTime=1766062998332, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1208519065233125464, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208518757253779608, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1208519065233125465, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1208518757253779608, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1174, endPage=1180, ext={EN=ArticleExt(id=1208518761552941296, articleId=1208518760273678561, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Effect of SE-hBCG on the immunogenicity of recombinant cytomegalovirus glycoprotein B and freeze-dried rabies vaccine for human use, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the effect of SE-hBCG on the immunogenicity of recombinant cytomegalovirus glycoprotein B (CgB) and freeze-dried rabies human vaccine (Rab). Methods A total of 145 SPF female BALB/c mice aged 6-8 weeks were selected for the following experiments. In experiment one, five groups of mice (five mice per group) received intramuscular injections of 0.2 ml of SE, SE-hBCG, CgB, SE+CgB or SE-hBCG+CgB three times (at week 0, 2 and 4), respectively.Two weeks after the last immunization was the endpoint of this experiment. In experiment two, four groups of mice (30 mice per group) received two intraperitoneally injections at week 0 and 1 with 0.2 ml of PBS, Rab, SE+Rab or SE-hBCG+Rab, respectively.Samples were collected at 4, 8, 10, 12, 14, 35 days after the first immunization. Spleen lymphocytes of mice were isolated after homogenizing spleens. The enzyme-linked immunosorbent spot assay (ELISPOT) was used to detect the number of antigen-specific IFN-γ and IL-4-secreting spot forming cells (IFN-γ-SFC or IL-4-SFC). Sera were harvested from eyeball blood. Antigen-specific IgG was detected using ELISA. Results SE-hBCG induced mice to produce CgB-specific Th1 responses as mice immunized with SE-hBCG produced greater numbers of IFN-γ-SFC than mice immunized with SE (266.0±87.9 vs. 104.5±28.8, P<0.05). Mice immunized with SE-hBCG adjuvanted CgB vaccines produced higher levels of CgB-specific IgG antibodies (18 800.0±2396.0) and greater numbers of IFN-γ-SFC (440.5±38.4) than mice immunized with CgB (3333.0±737.9 for CgB-specific IgG antibody titer and 189.2±21.4 for IFN-γ-SFC, P<0.05). Four days after the first immunization, SE or SE-hBCG adjuvanted Rab vaccines induced low-level anti-Rab IgG antibodies (OD450≤0.2). Anti-Rab IgG antibodies of mice immunized with Rab, SE or SE-hBCG adjuvanted Rab peaked 12 days after the first immunization. Mice immunized with SE-hBCG adjuvanted Rab vaccines produced higher levels of anti-Rab IgG antibodies 8, 10, 14, and 35 days (1700.0±200.0, 19 400.0±1661.3, 23 000±358.9, 23 600.0±6038.2, respectively)after the first immunization than mice immunized with Rab (310.0±97.9, 6730.0±1655.4, 6000.0±1655.6, 4400.0±1655.5,respectively, P<0.05) and greater numbers of IFN-γ-SFC 4, 8, 14 and 35 days (110.8±52.5, 213.0±29.7, 105.2±33.7,80.4±36.8, respectively) after the first immunization than mice immunized with PBS (6.4±3.5, 32.2±12.9, 11.4±5.1, 4.4±2.5,respectively, P<0.05). Conclusion SE-hBCG could enhance the immunogenicity of CgB and Rab, and it is likely to become an immunopotentiator.

, correspAuthors=Xiu-Yun He, authorNote=null, correspAuthorsNote=
*E-mail:
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目的 探讨含角鲨烯的水包油纳米乳剂-热灭活卡介苗(SE-hBCG)对重组巨细胞病毒糖蛋白B(CgB)及冻干人用狂犬病疫苗(Rab)免疫原性的影响。方法 选取145只SPF级6~8周龄雌性BALB/c小鼠进行以下实验。实验1:取25只小鼠,分成SE组、SE-hBCG组、CgB组、SE+CgB组、SE-hBCG+CgB组等5组,每组5只;于实验开始第0、2、4周分别给每只小鼠肌内注射SE、SE-hBCG、CgB、SE+CgB、SE-hBCG+CgB各0.2 ml,末次免疫后2周解剖小鼠。实验2:取120只小鼠,分成PBS组、Rab组、SE+Rab组、SE-hBCG+Rab组等4组,每组30只;于实验开始第0周及第1周每只小鼠分别腹腔注射PBS、Rab、SE+Rab、SE-hBCG+Rab各0.2 ml,于初免后第4、8、10、12、14、35天各解剖5只小鼠。无菌摘取小鼠脾脏,制备脾淋巴细胞悬液,采用酶联免疫斑点实验(ELISPOT)检测抗原特异的IFN-γ或IL-4的斑点形成细胞数(IFN-γ-SFC或IL-4-SFC);取小鼠眼球血,离心后收集血清,采用酶联免疫吸附试验(ELISA)检测抗原特异的IgG水平。结果 SE-hBCG组诱导小鼠产生CgB特异的IFN-γ-SFC(266.0±87.9)高于SE组(104.5±28.8,P<0.05);SE-hBCG+CgB组诱导小鼠产生抗-CgB IgG抗体滴度(18 800.0±2396.0)及CgB特异的IFN-γ-SFC(440.5±38.4)高于CgB组(抗体滴度为3333.0±737.9,IFN-γ-SFC为189.2±21.4,P<0.05)。SE+Rab或SE-hBCG+Rab初免后第4天可诱导低水平抗-Rab IgG抗体(OD450≤0.2);Rab、SE+Rab及SE-hBCG+Rab组抗-Rab IgG抗体均于初免后第12天达到峰值;SE-hBCG+Rab组于初免后第8、10、14及35天检测的抗-Rab IgG抗体(分别为1700.0±200.0、19 400.0±1661.3、23 000±358.9、23 600.0±6038.2)均高于Rab组(分别为310.0±97.9、6730.0±1655.4、6000.0±1655.6、4400.0±1655.5,P<0.05)。SE-hBCG+Rab组初免后第4、8、14及35天检测的Rab特异IFN-γ-SFC(分别为110.8±52.5、213.0±29.7、105.2±33.7、80.4±36.8)均高于对照组(分别为6.4±3.5、32.2±12.9、11.4±5.1、4.4±2.5,P<0.05)。结论 SE-hBCG可增强CgB及Rab的免疫原性,是一种较好的免疫增强剂。

, correspAuthors=何秀云, authorNote=null, correspAuthorsNote=
何秀云,E-mail:
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李廷慧,博士研究生,主要从事白癜风及感染性皮肤病方面的研究

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李廷慧,博士研究生,主要从事白癜风及感染性皮肤病方面的研究

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李廷慧,博士研究生,主要从事白癜风及感染性皮肤病方面的研究

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J Innate Immun, 2014, 6(2): 152-158., articleTitle=Long-lasting effects of BCG vaccination on both heterologous Th1/Th17 responses and innate trained immunity, refAbstract=null), Reference(id=1208518771686380149, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208518760273678561, doi=null, pmid=null, pmcid=null, year=2018, volume=172, issue=1-2, pageStart=176, pageEnd=190, url=null, language=null, rfNumber=[23], rfOrder=26, authorNames=Kaufmann E, Sanz J, Dunn JL, journalName=Cell, refType=null, unstructuredReference=Kaufmann E, Sanz J, Dunn JL, et al. BCG educates hematopoietic stem cells to generate protective innate immunity against tuberculosis[J]. 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SE. 含角鲨烯的水包油纳米乳剂;hBCG. 热灭活卡介苗;CgB. 巨细胞病毒糖蛋白B;与SE-hBCG组比较,(1)P<0.05;与CgB组比较,(2)P<0.05。

, figureFileSmall=+71+tXphCYxa3uqM9OHF9A==, figureFileBig=MBrWbNGEKqyslaidXmpv0A==, tableContent=null), ArticleFig(id=1208518766971982316, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208518760273678561, language=EN, label=Fig. 2, caption=CgB-specific IFN-γ-SFC (A) and IL-4-SFC (B) induced by different immunogens of mice, figureFileSmall=D6k4fHOmSr9f544BqRhfIA==, figureFileBig=20rf8YkhH/FhXmF1BLrljw==, tableContent=null), ArticleFig(id=1208518767060062705, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208518760273678561, language=CN, label=图2, caption=不同免疫原诱导小鼠CgB特异IFN-γ-SFC (A)及IL-4-SFC (B)的比较

SFC. 斑点形成细胞数;SE. 含角鲨烯的水包油纳米乳剂;hBCG. 热灭活卡介苗;CgB. 巨细胞病毒糖蛋白B;与SE组比较,(1)P<0.05;与SE-hBCG组比较,(2)P<0.05;与CgB组比较,(3)P<0.05;与SE+CgB组比较,(4)P<0.05。

, figureFileSmall=D6k4fHOmSr9f544BqRhfIA==, figureFileBig=20rf8YkhH/FhXmF1BLrljw==, tableContent=null), ArticleFig(id=1208518767160726005, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208518760273678561, language=EN, label=Fig. 3, caption=Influence of SE-hBCG on the anti-Rab IgG antibodies, figureFileSmall=pL/gpikEGGm+BolRdlgLjw==, figureFileBig=MmuVb6mQm27n7JHMUirtRQ==, tableContent=null), ArticleFig(id=1208518767273972216, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208518760273678561, language=CN, label=图3, caption=SE-hBCG对Rab诱导抗-Rab IgG抗体水平的影响

SE. 含角鲨烯的水包油纳米乳剂;hBCG. 热灭活卡介苗;Rab. 狂犬病疫苗;A. 初免后第4天各组抗体水平(OD450);B. 各组不同时间点抗体滴度比较(以PBS组血清为临界值);与PBS组比较,(1)P<0.05;与Rab组比较,(2)P<0.05;与SE+Rab组比较,(3)P<0.05。

, figureFileSmall=pL/gpikEGGm+BolRdlgLjw==, figureFileBig=MmuVb6mQm27n7JHMUirtRQ==, tableContent=null), ArticleFig(id=1208518767399801340, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208518760273678561, language=EN, label=Fig. 4, caption=Levels of Rab-specific IFN-γ-secreting spot forming cells of mice in each group after the first immunization, figureFileSmall=WARd0XNDNX3d2GGQb8dEHQ==, figureFileBig=W+3jG55nDOq8JH0EJHFtbQ==, tableContent=null), ArticleFig(id=1208518767483687428, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1208518760273678561, language=CN, label=图4, caption=各组小鼠初免后不同时间点Rab特异的IFN-γ-SFC水平比较

SE. 含角鲨烯的水包油纳米乳剂;hBCG. 热灭活卡介苗;Rab. 狂犬病疫苗;与PBS组比较,(1)P<0.05;与Rab组比较,(2)P<0.05;与SE+Rab组比较,(3)P<0.05。

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SE-hBCG对重组巨细胞病毒糖蛋白B及冻干人用狂犬病疫苗免疫原性的影响
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李廷慧 1 , 李彬钰 1 , 马锡惠 1 , 何秀云 1, 2, *
解放军医学杂志 | 基础研究 2021,46(12): 1174-1180
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解放军医学杂志 | 基础研究 2021, 46(12): 1174-1180
SE-hBCG对重组巨细胞病毒糖蛋白B及冻干人用狂犬病疫苗免疫原性的影响
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李廷慧1, 李彬钰1, 马锡惠1, 何秀云1, 2, *
作者信息
  • 1解放军总医院第八医学中心皮肤科,北京 100091
  • 2解放军总医院第三医学中心泌尿外科医学部,北京 100039
  • 李廷慧,博士研究生,主要从事白癜风及感染性皮肤病方面的研究

通讯作者:

何秀云,E-mail:
Effect of SE-hBCG on the immunogenicity of recombinant cytomegalovirus glycoprotein B and freeze-dried rabies vaccine for human use
Ting-Hui Li1, Bin-Yu Li1, Xi-Hui Ma1, Xiu-Yun He1, 2, *
Affiliations
  • 1Department of Dermatology, the Eighth Medical Center of Chinese PLA General Hospital, Beijing 100091, China
  • 2Department of Urology, the Third Medical Center of Chinese PLA General Hospital, Beijing 100039, China
出版时间: 2021-12-28 doi: 10.11855/j.issn.0577-7402.2021.12.02
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目的 探讨含角鲨烯的水包油纳米乳剂-热灭活卡介苗(SE-hBCG)对重组巨细胞病毒糖蛋白B(CgB)及冻干人用狂犬病疫苗(Rab)免疫原性的影响。方法 选取145只SPF级6~8周龄雌性BALB/c小鼠进行以下实验。实验1:取25只小鼠,分成SE组、SE-hBCG组、CgB组、SE+CgB组、SE-hBCG+CgB组等5组,每组5只;于实验开始第0、2、4周分别给每只小鼠肌内注射SE、SE-hBCG、CgB、SE+CgB、SE-hBCG+CgB各0.2 ml,末次免疫后2周解剖小鼠。实验2:取120只小鼠,分成PBS组、Rab组、SE+Rab组、SE-hBCG+Rab组等4组,每组30只;于实验开始第0周及第1周每只小鼠分别腹腔注射PBS、Rab、SE+Rab、SE-hBCG+Rab各0.2 ml,于初免后第4、8、10、12、14、35天各解剖5只小鼠。无菌摘取小鼠脾脏,制备脾淋巴细胞悬液,采用酶联免疫斑点实验(ELISPOT)检测抗原特异的IFN-γ或IL-4的斑点形成细胞数(IFN-γ-SFC或IL-4-SFC);取小鼠眼球血,离心后收集血清,采用酶联免疫吸附试验(ELISA)检测抗原特异的IgG水平。结果 SE-hBCG组诱导小鼠产生CgB特异的IFN-γ-SFC(266.0±87.9)高于SE组(104.5±28.8,P<0.05);SE-hBCG+CgB组诱导小鼠产生抗-CgB IgG抗体滴度(18 800.0±2396.0)及CgB特异的IFN-γ-SFC(440.5±38.4)高于CgB组(抗体滴度为3333.0±737.9,IFN-γ-SFC为189.2±21.4,P<0.05)。SE+Rab或SE-hBCG+Rab初免后第4天可诱导低水平抗-Rab IgG抗体(OD450≤0.2);Rab、SE+Rab及SE-hBCG+Rab组抗-Rab IgG抗体均于初免后第12天达到峰值;SE-hBCG+Rab组于初免后第8、10、14及35天检测的抗-Rab IgG抗体(分别为1700.0±200.0、19 400.0±1661.3、23 000±358.9、23 600.0±6038.2)均高于Rab组(分别为310.0±97.9、6730.0±1655.4、6000.0±1655.6、4400.0±1655.5,P<0.05)。SE-hBCG+Rab组初免后第4、8、14及35天检测的Rab特异IFN-γ-SFC(分别为110.8±52.5、213.0±29.7、105.2±33.7、80.4±36.8)均高于对照组(分别为6.4±3.5、32.2±12.9、11.4±5.1、4.4±2.5,P<0.05)。结论 SE-hBCG可增强CgB及Rab的免疫原性,是一种较好的免疫增强剂。

水包油纳米乳剂  /  卡介苗  /  巨细胞病毒糖蛋白B  /  狂犬病疫苗

Objective To investigate the effect of SE-hBCG on the immunogenicity of recombinant cytomegalovirus glycoprotein B (CgB) and freeze-dried rabies human vaccine (Rab). Methods A total of 145 SPF female BALB/c mice aged 6-8 weeks were selected for the following experiments. In experiment one, five groups of mice (five mice per group) received intramuscular injections of 0.2 ml of SE, SE-hBCG, CgB, SE+CgB or SE-hBCG+CgB three times (at week 0, 2 and 4), respectively.Two weeks after the last immunization was the endpoint of this experiment. In experiment two, four groups of mice (30 mice per group) received two intraperitoneally injections at week 0 and 1 with 0.2 ml of PBS, Rab, SE+Rab or SE-hBCG+Rab, respectively.Samples were collected at 4, 8, 10, 12, 14, 35 days after the first immunization. Spleen lymphocytes of mice were isolated after homogenizing spleens. The enzyme-linked immunosorbent spot assay (ELISPOT) was used to detect the number of antigen-specific IFN-γ and IL-4-secreting spot forming cells (IFN-γ-SFC or IL-4-SFC). Sera were harvested from eyeball blood. Antigen-specific IgG was detected using ELISA. Results SE-hBCG induced mice to produce CgB-specific Th1 responses as mice immunized with SE-hBCG produced greater numbers of IFN-γ-SFC than mice immunized with SE (266.0±87.9 vs. 104.5±28.8, P<0.05). Mice immunized with SE-hBCG adjuvanted CgB vaccines produced higher levels of CgB-specific IgG antibodies (18 800.0±2396.0) and greater numbers of IFN-γ-SFC (440.5±38.4) than mice immunized with CgB (3333.0±737.9 for CgB-specific IgG antibody titer and 189.2±21.4 for IFN-γ-SFC, P<0.05). Four days after the first immunization, SE or SE-hBCG adjuvanted Rab vaccines induced low-level anti-Rab IgG antibodies (OD450≤0.2). Anti-Rab IgG antibodies of mice immunized with Rab, SE or SE-hBCG adjuvanted Rab peaked 12 days after the first immunization. Mice immunized with SE-hBCG adjuvanted Rab vaccines produced higher levels of anti-Rab IgG antibodies 8, 10, 14, and 35 days (1700.0±200.0, 19 400.0±1661.3, 23 000±358.9, 23 600.0±6038.2, respectively)after the first immunization than mice immunized with Rab (310.0±97.9, 6730.0±1655.4, 6000.0±1655.6, 4400.0±1655.5,respectively, P<0.05) and greater numbers of IFN-γ-SFC 4, 8, 14 and 35 days (110.8±52.5, 213.0±29.7, 105.2±33.7,80.4±36.8, respectively) after the first immunization than mice immunized with PBS (6.4±3.5, 32.2±12.9, 11.4±5.1, 4.4±2.5,respectively, P<0.05). Conclusion SE-hBCG could enhance the immunogenicity of CgB and Rab, and it is likely to become an immunopotentiator.

oil-in-water nanomoemulsion  /  Bacille Calmette-Guerin vaccine  /  cytomegalovirus glycoprotein B  /  rabies vaccine
李廷慧, 李彬钰, 马锡惠, 何秀云. SE-hBCG对重组巨细胞病毒糖蛋白B及冻干人用狂犬病疫苗免疫原性的影响. 解放军医学杂志, 2021 , 46 (12) : 1174 -1180 . DOI: 10.11855/j.issn.0577-7402.2021.12.02
Ting-Hui Li, Bin-Yu Li, Xi-Hui Ma, Xiu-Yun He. Effect of SE-hBCG on the immunogenicity of recombinant cytomegalovirus glycoprotein B and freeze-dried rabies vaccine for human use[J]. Medical Journal of Chinese People’s Liberation Army, 2021 , 46 (12) : 1174 -1180 . DOI: 10.11855/j.issn.0577-7402.2021.12.02
卡介苗(Mycobacterium bovis Bacillus Calmette-Guérin,BCG)是目前唯一被批准用于预防结核病的疫苗。但对于感染结核杆菌(MTB)、患免疫缺陷症、先天及后天免疫不全的婴儿或儿童,临床上禁止接种BCG。热灭活BCG(hBCG)诱导的免疫应答弱于BCG,但仍具备BCG的生物学功能,且消除了MTB等分枝杆菌活菌产生的不良反应[1],其抗MTB感染的作用可被佐剂增强[2-3]。BCG的细胞壁、含CpG基序的核酸(BCG-CpG-DNA)、多糖等均可作用于不同的Toll样受体(toll-like recepter,TLR),从而启动天然免疫应答。因此,BCG、hBCG及其组分常作为疫苗佐剂,也可用于肺癌、恶性淋巴瘤根治手术或化疗后的辅助治疗[4-8]
1997年,意大利首次批准将含角鲨烯的MF59佐剂流感疫苗用于临床[9]。MF59通过TLR非依赖的MyD88信号通路启动或增强免疫应答,BCG可激活TLR-MyD88信号通路启动天然免疫,因此,MF59与hBCG可通过共同的MyD88信号通路产生协同作用。鉴于商品化的MF59价格偏贵,但其制备材料较便宜,本课题组前期制备了含角鲨烯的水包油纳米乳剂(SE)并开展研究,发现SE与hBCG具有协同作用,即SE可增强hBCG的免疫原性[10],而hBCG可增强SE募集巨噬细胞到注射部位的能力[11],将SE与hBCG结合制备的复合佐剂SE-hBCG能增强重组MTB蛋白特异的免疫应答[12]。目前,关于SE-hBCG对病毒抗原的辅佐效果仍不清楚,本研究探讨了SE-hBCG对天然及重组病毒蛋白免疫原性的影响,以进一步阐明SE-hBCG潜在的广谱佐剂特性。
6~8周龄SPF级雌性BALB/c小鼠145只(北京维通利华实验动物技术有限公司),体重18~20 g。冻干人用狂犬病疫苗(Rab,辽宁成大生物股份有限公司);小鼠干扰素-γ(IFN-γ)及白细胞介素(IL)-4酶联免疫斑点试剂盒(瑞典Mabtech公司);角鲨烯、Tween 20、刀豆蛋白A(CoA)及Span85(美国Sigma公司);优质胎牛血清及RPMI 1640培养基(美国Gibco公司);辣根过氧化物酶(HRP)标记的羊抗小鼠IgG二抗(美国Jackson ImmunoResearch Labtoratories公司);小鼠淋巴细胞分离液、0.5 mg/ml重组巨细胞病毒糖蛋白B(CgB原液)及广谱型酶联免疫斑点实验(ELISPOT)专用无血清培养基(北京达科为生物技术有限公司);即用型TMB显色液(杭州联科生物技术股份有限公司)。
SE及hBCG原液制备参照文献[12],1人份冻干Rab疫苗按说明书加入0.5 ml注射用水溶解,多人份溶解的Rab疫苗合并混匀即为Rab原液。
实验1免疫原制备:SE原液与等体积PBS混匀为SE,SE原液与等体积500 μg/ml的hBCG混匀为复合佐剂SE-hBCG;CgB原液用PBS稀释至25 μg/ml为CgB,CgB原液用PBS稀释至50 μg/ml后再与等体积SE混匀为SE+CgB;PBS稀释hBCG原液及CgB原液至终浓度分别为500 μg/ml与50 μg/ml的混合液,再与等体积SE混匀为SE-hBCG+CgB。实验2免疫原制备:0.01 ml Rab原液补加PBS至0.2 ml混匀为1/50人用剂量Rab(简称Rab),0.01 ml Rab原液补加PBS至0.1 ml,再与0.1 ml SE混匀为SE+Rab;0.1 ml Rab和hBCG的混合液(含0.01 ml Rab原液、终浓度为500 μg/ml的hBCG)与0.1 ml SE混匀为SE-hBCG+Rab。
实验1:取25只小鼠,分成5组:SE组、SE-hBCG组、CgB组、SE+CgB组、SE-hBCG+CgB组,每组5只;每只小鼠分别肌内注射SE、SE-hBCG、CgB、SE+CgB、SE-hBCG+CgB各0.2 ml,共免疫3次,每次免疫间隔2周;末次免疫后2周解剖小鼠。实验2:取120只小鼠,分成4组:PBS组、Rab组、SE+Rab组、SE-hBCG+Rab组,每组30只;每只小鼠分别腹腔注射PBS、Rab、SE+Rab、SE-hBCG+Rab各0.2 ml(各组Rab剂量均为1/50人用剂量),共免疫2次,每次免疫间隔1周;第1次免疫(初免)后4、8、10、12、14及35 d,各解剖5只小鼠。同时摘取小鼠眼球取血,离心收集血清用于抗体检测;无菌摘取脾脏制备脾淋巴细胞悬液(除初免后第10天及第12天解剖小鼠外),用于ELISPOT检测。实验过程符合国家及单位实验动物管理和使用的相关规定。
用包被液(0.05 mol/L碳酸盐缓冲液,pH 9.6)稀释CgB至5 μg/ml或1:300稀释Rab原液,每孔包被100 μl,4 ℃过夜;弃包被液,控干,每孔加入200 μl含0.05% Tween 20的PBS(PBST)洗板5 min,洗3次后控干。每孔加入200 μl含1%牛血清白蛋白的PBST(PBSTB),于37 ℃作用2 h。弃封闭液,控干,同上洗板并控干;每孔加入100 μl PBSTB稀释的血清(SE或PBS免疫小鼠血清1:100稀释,其血清按预实验确定稀释度,不少于3个),于37 ℃孵育2 h。弃血清,控干,同上洗板并控干;每孔加入100 μl PBSTB稀释的HRP标记的二抗(1:20 000),于37 ℃孵育1 h。弃二抗稀释液,控干,同上洗板;每孔加入TMB显色液100 μl,室温避光放置30 min,加入50 μl浓度为2 mol/L的H2SO4终止显色,于450 nm波长处测量光密度值(OD450)。以PBS(实验2)或SE(实验1)免疫小鼠血清1:100稀释检测OD4502 s作为临界值,其他免疫原免疫小鼠检测OD450大于临界值的最大稀释度的倒数作为抗体滴度。
按小鼠淋巴细胞分离液说明书制备脾淋巴细胞悬液并计数,用ELISPOT专用无血清培养基将脾淋巴细胞密度调整至2.5×106个/ml。取出预包被抗IFN-γ或IL-4单克隆抗体的96孔板,每孔加200 μl PBS(0.22 μm膜过滤且灭菌),洗板5次,控干,每孔加入200 μl含10%胎牛血清的RPMI 1640培养基,室温孵育30 min。弃去培养基,控干;将调整好的小鼠淋巴细胞悬液加入3孔中,100 μl/孔,然后分别加50 μl无血清培养基(阴性孔)、50 μl无血清培养基稀释的15 μg/ml CgB或Rab(原液按1:50稀释抗原孔)、50 μl无血清培养基稀释的15 μg/ml CoA(阳性孔)。将96孔板于37 ℃、含5%CO2的培养箱中培养36~40 h。取出微孔板,弃液体,每孔加200 μl PBS(0.22 μm膜过滤)洗板5次,控干,每孔加入100 μl检测抗体,室温孵育2 h。弃去孔内溶液,同上洗板5次,控干,每孔加入100 μl链霉亲和素-碱性磷酸酶结合物,室温孵育1 h。弃去孔内溶液,同上洗板5次,控干,加入100 μl 5-溴-4-氯-3-吲哚基-磷酸盐/四唑硝基蓝(BCIP/NBT)底物溶液(0.45 μm膜过滤),室温避光放置10~30 min,待出现斑点后,用蒸馏水洗涤各孔,将微孔板置通风处避光干燥。用酶联免疫斑点读板仪(美国CTL公司)读板,自动分析斑点形成细胞数(spot forming cells,SFC)。对阳性孔SFC>500的小鼠,计算抗原特异的分泌IFN-γ或IL-4的SFC(IFN-γ-SFC或IL-4-SFC)。抗原特异的IFN-γ-SFC或IL-4-SFC=抗原孔SFC—阴性孔SFC。
采用SPSS 16.0软件进行统计分析。正态分布的计量资料以$\bar{x}±s$表示,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。P<0.05为差异有统计学意义。
ELISA检测结果显示,与SE-hBCG组(419.7±61.2)及CgB组(3333.0±737.9)比较,SE+CgB组及SE-hBCG+CgB组小鼠血清抗-CgB IgG抗体滴度(分别为21 333.0±2186.0、18 800.0±2396.0)明显增加,差异有统计学意义(P<0.05,图1)。
ELISPOT检测结果显示,与SE组(104.5±28.8)比较,SE-hBCG组、SE-hBCG+CgB组CgB特异的IFN-γ-SFC(分别为266.0±87.9、440.5±38.4)均明显增加,差异有统计学意义(P<0.05);与SE-hBCG+CgB组(440.5±38.4)比较,SE-hBCG组、CgB组、SE+CgB组CgB特异的IFN-γ-SFC(分别为266.0±87.9、189.2±21.4、199.2±38.6)均明显减少,差异有统计学意义(P<0.05,图2A)。与CgB组(149.0±45.8)比较,SE组、SE-hBCG组、SE+CgB组、SE-hBCG+CgB组CgB特异的IL-4-SFC(分别为59.0±14.5、14.0±13.0、56.0±35.9、31.2±11.3)均明显减少,差异有统计学意义(P<0.05,图2B)。
初免后第4天,与PBS组及Rab组(OD450分别为0.094±0.006、0.096±0.003)比较,SE+Rab组及SE-hBCG+Rab组抗-Rab IgG抗体增加(OD450分别为0.20±0.01、0.15±0.01),差异有统计学意义(P<0.05,图3A)。
初免后第8~35天,Rab组、SE+Rab组、SE-hBCG+Rab组免疫小鼠产生抗-Rab IgG抗体滴度随时间变化趋势基本一致,均于初免后第12天达到峰值(图3B);其中Rab组初免后第12天的抗-Rab IgG抗体滴度(16 200.0±6740.9)高于初免后第8、1 0、1 4、3 5天(分别为310.0±97.9、6730.0±1655.4、6000.0±1655.6、4400.0±1655.5),差异有统计学意义(P<0.05);SE+Rab组初免后第12天的抗-Rab IgG抗体滴度(36 000.0±6595.5)高于初免后第8、10、14、35天(分别为2000.0±273.9、7600.0±979.8、23 000.0±4358.9、12 000.0±2708.0),差异有统计学意义(P<0.05),且初免后第14天的抗-Rab IgG抗体滴度高于初免后第8、10天(P<0.05);SE-hBCG+Rab初免后第12天的抗-Rab IgG抗体滴度(37 000.0±6633.2)高于初免后第8、10、14、35天(分别为1700.0±200.0、19 400.0±1661.3、23 000.0±358.9、23 600.0±6 038.2),差异有统计学意义(P<0.05),且初免后第8天的抗-Rab IgG抗体滴度低于初免后第10、12、14、35天,差异有统计学意义(P<0.05)。
同一时间不同组抗-Rab IgG抗体滴度比较结果显示,初免后第8、14天,SE+Rab组及SE-hBCG+Rab组抗-Rab IgG抗体滴度均高于Rab组(P<0.05);初免后第10天,SE-hBCG+Rab组抗-Rab IgG抗体滴度高于Rab组及SE+Rab组(P<0.05);初免后第35天,SE-hBCG+Rab组抗-Rab IgG抗体滴度高于Rab组,差异有统计学意义(P<0.05,图3B)。
不同时间组内比较,Rab组、SE+Rab组及SE-hBCG+Rab组诱导Rab特异的IFN-γ-SFC随时间变化趋势基本一致,即先升高后降低(图4)。其中Rab组初免后第8天Rab特异的IFN-γ-SFC达到峰值(243.6±83.3),明显高于初免后第4、14及35天(分别为44.6±14.1、61.4±18.2、48.8±7.0),差异有统计学意义(P<0.05);SE+Rab组初免后第14天的Rab特异的IFN-γ-SFC达到峰值(250.6±19.3),明显高于初免后第4、8及35天(分别为91.6±37.1、109.0±39.4、45.2±16.9),差异有统计学意义(P<0.05);SE-hBCG+Rab组初免后第8天的Rab特异的IFN-γ-SFC达到峰值(213.0±29.7),明显高于初免后第35天(80.4±36.8),差异有统计学意义(P<0.05,图4)。
同一时间组间比较:初免后第4天,与对照组(6.4±3.5)比较,SE-hBCG+Rab组Rab特异的IFN-γ-SFC(110.8±52.5)明显增加,差异有统计学意义(P<0.05);初免后第8天,与对照组(32.2±12.9)比较,Rab组、SE-hBCG+Rab组Rab特异的IFN-γ-SFC(分别为243.6±83.3、213.0±29.7)明显增加,差异有统计学意义(P<0.05);初免后第14天,与SE+Rab组(250.6±19.3)比较,对照组、Rab组、SE-hBCG+Rab组Rab特异的IFN-γ-SFC(分别为11.4±5.1、61.4±18.2、105.2±33.7)明显减少(P<0.05),且与对照组比较,SE-hBCG+Rab组Rab特异的IFN-γ-SFC明显增加(P<0.05);初免后第35天,与对照组(4.4±2.5)比较,SE-hBCG+Rab组Rab特异的IFN-γ-SFC(80.4±36.8)明显升高,差异有统计学意义(P<0.05,图4)。
重组蛋白疫苗可高产、高纯度、低成本且安全性更好,但单纯重组蛋白的免疫原性一般较差,需要佐剂增强机体对抗原的免疫应答或改变免疫应答类型。本研究结果显示,SE-hBCG作为重组蛋白CgB佐剂诱导的抗-CgB IgG抗体滴度与SE+CgB组相当,但抗原特异的IFN-γ-SFC明显高于单纯CgB组及SE+CgB组,而抗原特异的IL-4-SFC明显低于单纯CgB组,表明SE-hBCG作为重组蛋白CgB佐剂有利于细胞免疫应答偏向Th1型。进入临床研究的MF59佐剂CgB疫苗的免疫原性优于氢氧化铝佐剂CgB疫苗[13],但免疫保护效果不理想[14-15];这是因为该疫苗不能诱导Th1型免疫反应[16]。本研究结果显示CgB主要诱导抗原特异的IL-4-SFC,而文献报道MF59佐剂疫苗肌内注射诱导的免疫反应一般以Th2型免疫为主[17]。因此,通过分析SE、SE-hBCG及MF59作为CgB佐剂诱导的免疫原性特征,推测SE-hBCG佐剂CgB疫苗的免疫保护效果优于MF59佐剂CgB疫苗及SE佐剂疫苗。
SE-hBCG除了可增强病毒重组蛋白的免疫原性外,还可影响病毒天然蛋白的免疫原性。本研究结果显示,SE-hBCG作为狂犬病疫苗的佐剂,可使机体产生的抗原特异IgG及IFN-γ-SFC免疫应答提前且维持时间较长;除初免后第12天外的其他检测时间点,SE-hBCG佐剂Rab疫苗组抗原特异的IgG抗体滴度均高于单纯狂犬病疫苗组。有研究将BCG-CpG-DNA作为不同剂量狂犬病疫苗的佐剂免疫小鼠,结果发现血清中和抗体及抗原特异的IFN-γ-SFC及IL-2-SFC均显著高于单纯狂犬病疫苗组[18],与本研究结果基本一致。综合以上结果,笔者认为研制佐剂狂犬病疫苗可降低狂犬病疫苗剂量,减少接种针次,提早产生免疫应答,诱导远期免疫保护。
SE-hBCG诱导了CgB特异的Th1型免疫反应,但本实验缺乏hBCG免疫小鼠组,因此无法解释SE-hBCG诱导的CgB特异Th1型免疫反应是hBCG的作用还是SE与hBCG协同作用的结果。这种现象与BCG免疫训练产生的效果相似,即BCG可通过刺激固有免疫细胞使其产生免疫记忆,从而产生针对MTB及其他病原体的免疫反应和(或)免疫保护[19-23]。复合佐剂SE-hBCG通过激活天然免疫应答而增强抗原特异的免疫应答,也可通过免疫训练产生CgB蛋白特异的Th1型免疫反应,这为SE-hBCG作为广谱性佐剂提供了理论基础。此外,本实验仅验证了两种病毒抗原,还需要验证多种类型的抗原来阐明SE-hBCG复合佐剂的广谱性。
综上所述,SE-hBCG可增强狂犬病疫苗及重组蛋白CgB的免疫原性,包括增加抗原特异的IgG抗体生成并偏向Th1型免疫应答。由于本实验检测的免疫指标偏少,尚需要增加免疫指标以全面阐明SE-hBCG复合佐剂增强病毒抗原免疫应答的机制。
  • 北京市自然科学基金(7172212)
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2021年第46卷第12期
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doi: 10.11855/j.issn.0577-7402.2021.12.02
  • 接收时间:2021-03-29
  • 首发时间:2025-12-18
  • 出版时间:2021-12-28
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  • 收稿日期:2021-03-29
  • 修回日期:2021-08-09
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Natural Science Foundation of Beijing(7172212)
北京市自然科学基金(7172212)
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    1解放军总医院第八医学中心皮肤科,北京 100091
    2解放军总医院第三医学中心泌尿外科医学部,北京 100039

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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