陈梦娇,杨晓蕾,刘龙丁,郑惠文,罗娜,尹晓晓,李洪哲,邓燕,李在晗,孙明波,杨净思
CHEN Meng-jiao,YANG Xiao-lei,LIU Long-ding,ZHENG Hui-wen,LUO Na,YIN Xiao-xiao,LI Hong-zhe,DENG Yan,LI Zai-han,SUN Ming-bo,YANG Jing-si

• 1:中国医学科学院北京协和医学院医学生物学研究所生物制品四室

摘要(Abstract)：

目的初步评价肠道病毒71型(enterovirus 71,EV71)与乙型肝炎(简称乙肝)病毒(hepatitis B,HBV)联合疫苗中两种抗原之间的相互作用及其免疫小鼠诱导的特异性免疫应答水平。方法将EV71灭活疫苗抗原和重组HBV疫苗抗原(HBs Ag)按常规剂量,采用直接吸附法吸附Al(OH)3佐剂,配制成EV71-HBV联合疫苗,并设单苗组、佐剂对照组和空白对照组(PBS),分别于0、28 d各经腓肠肌免疫BALB/c小鼠1次,于初免后第2、4周和第2剂免疫后第2、4、8周经尾动脉采血,分离血清,采用双抗体夹心ELISA法检测血清中Anti-HBs水平,微量中和试验检测血清中EV71中和抗体效价;并于初免后第3、4周和第2剂免疫后第1、2、4周取小鼠脾脏,分离脾单个核细胞(mononuclear cell,MNC),采用酶联免疫斑点试验(enzyme-linked immunospot assay,ELISPOT)检测MNC中抗原特异性IFNγ的分泌水平。结果第2剂免疫后第2周,各疫苗组小鼠血清EV71中和抗体和Anti-HBs阳转率均达100%,各检测时间点疫苗组间抗体效价差异均无统计学意义(P>0.05),佐剂对照组和空白对照组抗体均无阳转。各疫苗组分泌IFNγ的斑点形成细胞(spot forming cell,SFC)数在5个检测时间点有所波动,第2剂免疫后第1周,所有疫苗组IFNγ分泌水平均明显升高,达5个检测时间点的峰值,第2剂免疫后第2周显著下降,但在第2剂免疫后第4周又小幅升高;除第2剂免疫后第2周EV71-HBV联合疫苗组SFC数显著高于EV71单苗组外(P<0.05),其他检测时间点EV71-HBV联合疫苗组与单苗组的SFC数差异均无统计学意义(P>0.05);佐剂对照组和空白对照组的ELISPOT检测结果均为阴性。结论一定剂量的EV71和HBV联合疫苗可诱导小鼠产生良好的体液免疫和细胞免疫应答,两种抗原间未发现相互作用。
Objective To primarily evaluate the interaction of two antigens in enterovirus 71(EV71) and hepatitis B combined vaccine as well as the specific immune response level induced by the vaccine in mice. Methods EV71 and HBV combined vaccine(EV71-HBV) was formulated by direction adsorption of inactivated EV71 vaccine antigen and HBs Ag, at routine dosages, onto aluminum hydroxide adjuvant, with which BALB / c mice were immunized i.m. twice on days 0 and 28 respectively, using monovalent EV71 and HB vaccines, adjuvant and PBS(blank) as controls. Serum samples were collected at weeks 2 and 4 after the first dose and weeks 2, 4, and 8 after the second dose, and determined for anti-HBs level by double antibody sandwich ELISA and for neutralizing antibody titer against EV71 by micro-neutralization test. The spleens of mice were taken out at weeks 3 and 4 after the first dose and weeks 1, 2 and 4 after the second dose, from which mononuclear cells(MNCs) were isolated and determined for the secretion level of IFNγ by enzyme-linked immunospot assay(ELISPOT). Results All the positive conversion rates of neutralizing antibody against EV71 and anti-HBs of mice in various vaccine groups reached 100% at week 2 after the second dose, and the antibody titers at various time points showed no significant difference(P > 0. 05), while there was no seroconversion of antibody in adjuvant and blank control groups. The numbers of spot forming cells(SFCs) secreting IFNγ in various vaccine groups varied at five different times, and IFNγ secretion levels increased significantly at week 1 after the second dose and reached the peak value, then decreased significantly at week 2 and increased slightly at week 4 after the second dose. The number of SFCs in EV71-HBV group at week 2 after the second dose was significantly higher than that in EV71 vaccine group(P < 0. 05), while showed no significant difference with those in monovalent vaccine groups at other time points(P >0. 05). Both the ELISPOT results in adjuvant and blank control groups were negative. Conclusion EV71 and hepatitis B combined vaccine at a certain dosage induced high humoral and cellular immune responses in mice, and no interactions were observed between the two vaccine antigens.

关键词(KeyWords)： 肠道病毒71型;乙型肝炎病毒;联合疫苗;体液免疫;细胞免疫
Enterovirus 71(EV71);Hepatitis B virus;Combined vaccine;Humoral immunity;Cellular immunity

Abstract:

Keywords:

基金项目(Foundation): 国家十二五“重大新药创制”科技重大专项(2015ZX09101-031)

作者(Author): 陈梦娇,杨晓蕾,刘龙丁,郑惠文,罗娜,尹晓晓,李洪哲,邓燕,李在晗,孙明波,杨净思
CHEN Meng-jiao,YANG Xiao-lei,LIU Long-ding,ZHENG Hui-wen,LUO Na,YIN Xiao-xiao,LI Hong-zhe,DENG Yan,LI Zai-han,SUN Ming-bo,YANG Jing-si

DOI: 10.13200/j.cnki.cjb.000840

参考文献(References)：

• [1]Li R,Liu L,Mo Z,et al.An inactivated enterovirus 71 vaccine in healthy children[J].N Engl J Med,2014,370(9):829-837.
• [2]Solomon T,Lewthwaite P,Perera D,et al.Virology,epidemiology,pathogenesis,and control of enterovirus 71[J].Lancet Infect Dis,2010,10(11):778-790.
• [3]WHO Regional Office for the Western Pacific.A guide to clinical management and public health response for hand,foot and mouth disease(HFMD)[S].Manila:World Health Organization Western Pacific Region,2011.
• [4]Xing W,Liao Q,Viboud C,et al.Hand,foot,and mouth disease in China,2008-2012:an epidemiological study[J].Lancet Infect Dis,2014,14(4):308-318.
• [5]Zhou L,Ma FB,Tang FY,et al.Epidemic characteristics of hepatitis B after hepatitis B vaccine integrated into EPI[J].Chin J Public Health,2010,26(4):444-446.(in Chinese)周羚,马福宝,汤奋扬,等.乙肝疫苗免疫规划对乙肝流行影响[J].中国公共卫生,2010,26(4):444-446.
• [6]Wang LC,Hou QM,Zhang SM.Progress in research on combined vaccine home and abroad[J].Chin J Biologicals,2012,25(4):516-519.(in Chinese)王丽婵,侯启明,张庶民.国内外联合疫苗的研究新进展[J].中国生物制品学杂志,2012,25(4):516-519.
• [7]Li J,Zhang D,Ma R,et al.Preclinical evaluation of a twodose vaccination schedule of recombinant Hansenula polymorpha hepatitis B vaccine in animals[J].Hum Vaccin Immunother,2013,9(4):736-743.
• [8]Liang ZL.Standardization of methodology for the efficacy evaluation of hepatitis B vaccine[J].Chin J Viral Dis,2011,1(5):344-347.(in Chinese)梁争论.乙型肝炎疫苗免疫效果评价的方法学规范化考虑[J].中国病毒病杂志,2011,1(5):344-347.
• [9]Zeng C,Mawhinney S,Barón AE,et al.Evaluating ELISPOT summary measures with criteria for obtaining reliable estimates[J].J Immunol Methods,2005,297(1-2):97-108.
• [10]Sanchez AM,Rountree W,Berrong M,et al.The External Quality Assurance Oversight Laboratory(EQAPOL)proficiency program for IFN-gamma enzyme-linked immunospot(IFN-γELISpot)assay[J].J Immunol Methods,2014,409(1):31-43.
• [11]He W.Medical Immunology[J].2nd ed.Beijing:People's Medical Publishing House,2010:181-182.(in Chinese)何维.医学免疫学[M].2版.北京:人民卫生出版社,2010:181-182.
• [12]Kim M,Moon HB,Kim K,et al.Antigen dose governs the shaping of CTL repertoires in vitro and in vivo[J].Int Immunol,2006,18(3):435-444.
• [13]Ong KC,Badmanathan M,Devi S,et al.Pathologic characterization of a murine model of human enterovirus 71 encephalomyelitis[J].J Neuropathol Exp Neurol,2008,67(6):532-542.
• [14]Liu L,Zhao H,Zhang Y,et al.Neonatal rhesus monkey is a potential animal model for studying pathogenesis of EV71infection[J].Virology,2011,412(1):91-100.
• [15]Sa-Nguanmoo P,Rianthavorn P,Amornsawadwattana S,et al.Hepatitis B virus infection in non-human primates[J].Acta Virol,2009,53(2):73-82.