全球乙型肝炎疫苗研发分析

世界上大多数国家都制定了乙型肝炎(乙肝)免疫计划,这为预防性乙肝疫苗的研发提供了导向.从第一代血源性疫苗到重组乙肝疫苗,再到合成肽疫苗和DNA疫苗,乙肝疫苗的研制技术不断进步,其应用范围也已从预防扩展到治疗.本文分析了各国乙肝免疫计划与乙肝疫苗研发的关系,总结了预防性和治疗性乙肝疫苗的研发思路,并提出乙肝疫苗进一步研发的方向.     

乙型肝炎转基因小鼠品系C57-TgN(HBV adr2.0)SMMU的生物学特征

目的：评价乙肝转基因小鼠品系C57－TgN(HBV adr2.0)SMMU生物学特征的稳定性。方法：以F6代乙肝转基因小鼠C57－TgN(HBV adr2.0)SMMU为研究对象，采用基因组DNA PCR，血清ELISA检测，Western印迹分析，免疫组织化学，血清DNA PCR，透射电镜和H－E染色的方法分析HBV基因在转基因小鼠中的整合，表达，复制和组织这变化。结果：F6代乙肝转基因小鼠基因组中稳定整合有HBV基因，肝组织中可检测用HBsAg,HBcAg和X蛋白3种病毒蛋白，血清中HBsAg和HBeAg的表达率分别为19．54％和3．39％，且在血清和肝组织中存在病毒NA和病毒样颗粒；长期的病毒DNA整合，表达和复制可以引起转基因小鼠肝，肺等组织的病理性损伤。结论：乙肝转基因小鼠品系C57－TgN(HBV adr2.0)SMMU具有基因组中稳定整合病毒DNA，血清和肝组织中有病毒蛋白表达和病毒复制的特征，并具有一定的组织这变化，作为生物医药研究的实验动物模型具有推广应用价值。     

Comparative immunogenicity of plasma and recombinant hepatitis B virus vaccines in homosexual men

A randomized, double-blind clinical trial of plasma-derived and DNA recombinant hepatitis B virus vaccines was conducted in 186 homosexual men. Nine months after the immunization series (three doses) began, the seroconversion rate in the plasma vaccine group was 88% (68/77); this was significantly higher than the 74% (60/81) response rate of the recombinant vaccine group. Men positive for antibody to the human immunodeficiency virus (HIV) had a considerably higher nonresponse rate to either vaccine than expected in non-HIV-infected homosexual men. The odds ratios of nonresponse to hepatitis B virus vaccine for HIV-seropositive vs HIV-seronegative subjects were 12.0 (95% confidence interval, 1.7 to 89.3) and 13.6 (95% confidence interval, 2.3 to 148.3) for the plasma and DNA recombinant vaccines, respectively.     

HBV转基因在C57-TgN(HBVadr2.0)SMMU3小鼠基因组中的整合分析

目的:分析HBV转基因在乙肝转基因小鼠C57 TgN(HBVadr2.0)SMMU"3号"品系基因组中的整合特征.方法:以F6～F17代乙肝转基因小鼠C57 TgN(HBVadr2.0)SMMU为研究对象,采用基因组DNA PCR、Southern印迹和DNA测序的方法,分析HBV转基因在该品系转基因小鼠基因组中的整合特征.结果:PCR分析显示,F6～F17代乙肝转基因小鼠基因组中均已稳定整合了相同拷贝数的HBV转基因,整合的HBV DNA可通过生殖系在世代间稳定遗传.整合在转基因小鼠基因组中的HBV DNA含有HBVpres、s、c、x基因,Southern印迹证实HBV转基因含有全长HBV基因组DNA,DNA测序结果表明,该品系转基因小鼠所整合的转基因为adr亚型的HBV DNA.结论:C57-TgN(HBVadr2.0)SMMU"3号"品系转基因小鼠基因组中均整合有adr亚型的全长HBV基因组DNA,从DNA水平证实该转基因小鼠品系已具备了乙肝实验动物模型的基本条件.     

Safety evaluation in mice of the childhood immunization vaccines from two south-eastern states of Nigeria

Objective:To check the effects of the vaccines on the hematopoietic system and weight of mice after immunization.Methods:The study was done with the Expanded Programme on Immunization vaccines donated by the Ministries of Health of Abia and Imo States of Nigeria.The vaccines were collected from the cold-chain stores and transported in vaccine carriers to the cold-chain facility in Nnamdi Azikiwe University Teaching Hospital within 3 hours of collection.They were used to immunize a total of 160 mice.The Ethics Committee of Nnamdi Azikiwe University Teaching Hospital,Nnewi of Anambra State,Nigeria approved the protocol.Results:Mice body weight changes test showed that the mice all had increased body weight at Days 3 and 7 post-immunization and none died during the 7 d post-immunization observation.The percentage weight gains of the mice compared with the control were 69%.70%,64%.63%,65%and 68%for oral polio vaccine,diphtheria-pertussis-tetanus.bacillus CalmetteGuerin,measles,yellow fever and hepatitis B vaccines respectively collected from Imo State.The mice immunized with oral polio vaccine,pentavalent.bacillus Calmette-Guerin.measles,yellow fever and hepatitis B vaccines collected from Abia State had 123%.114%,121%.116%,142%and 119%weight gain respectively compared with the control.Leukocytosis promoting toxicity test showed that none of the vaccines was able to induce proliferation of leukocytes up to ten folds.Leukopenic toxicity test showed that all the vaccines had an leukopenic toxicity test value higher than 80%of the control(physiological saline).Conclusions:The vaccine samples tested were safe and did not affect the hematopoietic system adversely.The storage conditions of the vaccines in the States' cold-chain stores had not compromised the safety of the vaccines.     

乙肝表面抗原核酸疫苗诱导小鼠产生母源抗体

目的　研究核酸疫苗激发的抗体传递给仔代的能力。方法　以编码人类乙型肝炎病毒表面抗原 (ayw亚型 )的质粒 pcDNA HBs作为疫苗 ,肌肉注射 6周龄的BALB/c雌鼠 ,三星期后加强免疫一次 ,再过两周后使其配种怀孕 ,怀孕第 19天剖腹产取出胎儿并分离孕鼠及胎儿的血清 ,作ELISA检测。结果　经ELISA检测 ,10只小鼠中有 7只产生了抗体 (IgG)并传递给胎儿 ,且母体与胎儿的抗体效价相同 ,实验组与对照组差异显著 (P <0 .0 5 )。 结论　核酸疫苗与传统疫苗一样能诱导产生母源抗体。     

佐剂CpG ODN对HBV基因疫苗诱导抗体产生的影响

目的 探讨人工合成的ＣｐＧ ＯＤＮ作为佐剂对乙肝病毒基因疫苗诱导小鼠产生抗ＨＢｓ的影响。方法 构建编码乙型肝炎病毒（ＨＢＶ）表面抗原蛋白Ｓ的重组真核表达质粒ｐＣＲ３．１－Ｓ作为ＨＢＶ基因疫苗,人工合成含ＣｐＧ ｍｏｔｉｆ的硫代磷酸寡核苷酸作为佐剂,以ＢＡＬＢ／ｃ小鼠作为实验动物进行免疫接种,采用ＥＬＩＳＡ法检测免疫小鼠的抗ＨＢｓ应答。结果 与生理盐水作为佐剂组相比较,应用ＣｐＧ ＤＯＮ组小鼠产生更     

磷酸钙及不完全弗氏佐剂与乙肝核酸疫苗混合免疫小鼠的研究

目的：观察磷酸钙与不完全弗氏佐剂混合作为乙肝核酸疫苗的佐剂能否提高乙肝核酸疫苗的免疫效果。方法：将乙肝核酸疫苗与磷酸钙形成共沉物,再与不完全弗氏佐剂混合免疫小鼠,不同时间内观察小鼠外周血中ＨＢｓＡｇ与抗一ＨＢＳ的变化,从而观察小鼠的免疫应答。结果：两种佐剂能提高乙肝核酸疫苗的免疫效果。结论：核酸疫苗免疫中使用佐剂值得进一步研究。     

白细胞介素2增强HBV基因疫苗的免疫效应

目的：构建乙型肝炎病毒基因疫苗ｐＣＲ３．１－Ｓ,观察重组人白细胞介素２（ｒｈＩＬ－２）作为佐剂对其诱导ＮＡＬＢ／ｃ小鼠产生免疫应答的影响．方法：以ＥＬＩＳＡ法检测免疫小鼠血清抗ＨＢｓ,另用＾３Ｈ－ＴｄＲ参入法测定淋巴细胞增殖活性,初步研究不同免疫组的体液和细胞免疫应答．结果：ｒｈＩＬ－２组免疫小鼠抗ＨＢｓ抗体和淋巴细胞增殖活性与对照组相比有显著差异（Ｐ〈０．０５）．结论：ＨＢＶ基因疫苗可诱发ＢＡＬ     

基因乙肝疫苗接种母鸡的体液免疫应答

目的：探讨基因乙肝疫苗对母鸡免疫效果的影响。方法：用基因乙肝疫苗辅以佐剂皮下注射母鸡，随后加强免疫4次，用ELISA方法检测血清中抗－HBs。结果：在初次免疫母鸡血清中检出低滴度抗体，加强免疫后，抗体水平显著上升，滴度高达1：640，并维持较长时间，其抗体水平及抗体应答持久性与注射次数与注射途径有关。此外，用乙肝疫苗加佐剂比单用乙肝疫苗免疫效果明显要好。结论：应用基因乙肝疫苗能够诱导母鸡产生针对HBsAg的特异性体液免疫应答。     

乙型肝炎病毒核心抗原及Flt3配体双表达核酸疫苗的构建与体外表达

目的:构建可同时表达乙型肝炎病毒核心抗原(HBcAg)和Flt3配体(FL)胞外段的双表达核酸疫苗。方法:将HBcAg和FL胞外段的基因用PCR方法扩增并分别引入相应的限制性内切酶酶切位点,然后克隆入真核双表达载体pIRES,获得含双基因片段的表达质粒;再取该表达质粒中的内部核糖体切入位点(IRES)序列和基因片段克隆入pJW4303载体中,获得相应的双表达核酸疫苗,经筛选鉴定后,以293T细胞瞬时表达检测两基因的表达水平。结果:构建成功以pIRES、pJW4303为载体的4种HBcAg和FL胞外段的双表达核酸疫苗,体外表达证实HBcAg和FL胞外段均能高效表达,基因位于IRES上游时表达水平较高。结论:用IRES元件实现了HBcAg和FL胞外段双表达核酸疫苗的构建,pJW4303为载体的核酸疫苗两基因表达水平优于pIRES为载体的核酸疫苗。     

Recent advances in the identification of hepatitis viruses.

Resulting directly from the discovery of virus-related antigens, rapid progress has marked the last decade of viral hepatitis research. The hepatitis B virion has been tentatively identified as a DNA virus with an endogenous DNA polymerase, and new serological markers for type B hepatitis have been discovered. Hepatitis A antigen has been identified on a virus-like particle thought to be the hepatitis A virion. Progressively more sophisticated assays for hepatitis antigens and antibodies have been applied to the study of viral hepatitis epidemiology and biochemical-biophysical characterization of the agents. Most recently, knowledge learned from such studies has been exploited to develop a prototype non-infectious but immunogenic hepatitis B vaccine using hepatitis B surface antigen (HBsAg) purified in large quantities from chronic HBsAg carriers. Especially exciting is the prospect, suggested by serological studies of viral hepatitis, that hepatitis viruses besides hepatitis A and B viruses will be identified.     

含CpG基序的寡核苷酸对乙型肝炎病毒基因疫苗的细胞免疫效应的影响

目的探讨人工合成的CpG ODN作为佐剂对乙肝病毒基因疫苗诱导小鼠产生细胞免疫应答的影响.方法构建编码乙型肝炎病毒(HBV)表面抗原蛋白S的重组真核表达质粒pCR3.1-S作为HBV基因疫苗,人工合成含CpG基序(motif)的硫代磷酸寡核苷酸(CpG ODN)作为佐剂,观察其对HBV基因疫苗接种Balb/c小鼠诱导产生淋巴细胞增殖和淋巴细胞杀伤等细胞免疫应答的影响.结果与空载体对照组相比较,HBV基因疫苗诱发Balb/c小鼠产生较好的HBV特异的淋巴细胞增殖及杀伤效应(P＜0.05);应用CpGODN组与未应用组相比较,免疫小鼠产生更强的HBV特异性细胞免疫应答(P＜0.05).结论CpG ODN作为佐剂,对HBV基因疫苗诱导Balb/c小鼠产生细胞免疫应答具有促进作用.     

Detection of hepatitis B virus DNA in mononuclear blood cells

The Southern transfer hybridisation technique was used to test mononuclear blood cells for hepatitis B virus DNA. Viral DNA sequences were detected in mononuclear cells of 10 out of 16 patients with hepatitis B virus infection and in none of 21 normal controls. Blood contamination was excluded by the absence of hepatitis B virus DNA in the corresponding serum samples in all cases. Free monomeric hepatitis B virus DNA was found in three patients positive for hepatitis Be antigen (HBeAg) and one positive for anti-HBe, and integrated hepatitis B virus DNA was present in four patients positive for anti-HBe. In two other patients the small size of the samples did not allow a distinction between free and integrated viral DNA. The state of the virus in the mononuclear cells seemed to correlate with the HBeAg or anti-HBe state, as has been noted in the liver. These results indicate that hepatitis B virus may infect mononuclear blood cells, thereby expanding the tissue specificity of this agent beyond the liver, as has been reported for pancreatic, kidney, and skin tissue. They also suggest that hepatitis B virus infection of mononuclear cells might be related to immunological abnormalities observed in carriers of the virus.     

11166例病毒性肝炎流行病学统计分析

目的分析某地病毒性肝炎发病特征,为制订病毒性肝炎预防与控制措施提供依据。方法对甲、乙型和其他型肝炎进行流行病学统计分析。结果病毒性肝炎发病呈逐年上升趋势,没有季节聚集性,发病年龄以青壮年为主,农民、学生发病占病毒性肝炎总数的78.12%。男性发病多于女性,男女比为2.13:1,各年度男女发病无差异。结论有计划地在重点人群开展甲肝、乙肝疫苗的普种和加强接种为主导的综合性防制措施是控制甲、乙型肝炎疫情发生与流行的有效措施。同时加强儿童乙肝疫苗的基础免疫工作。     

FIFTEEN YEARS RESEARCH ON HEPATITIS B

This is a report on the laboratory research carried out by the Institute on hepatitis B during the last 15 years. The main aspects of the research were:1). Research on HBV. We successfully purified HBsAg by means of zonai density gradient ultracentrifugation; corresponding hepatitis B core antibody was obtained for immune animals, followed by the establishment of tbe technlque of HBcAg, Anti-HBc specific aad sensitive determination. Utilizing endogenous DNA polymerase reaction, 32P HBV DNA probe was made, which, with its specificity, can be used to detect and obtain pg Ievel directly.2). Research on immunochemistry in which the first lot of hepatitis B vaccine and HBIG was prepared in China.3). Research on applicatlon. Satisfactory results were achieved in the use of vaccines for blocking mother-infant transmission and in the use of prophylaxis for HBV infection among specialiy susceptible population, such as athletes on national teams.As outstanding works of scientific research, altogether 20 items hav     

Antibody response to E2 glycoprotein induced in mice by immunization with plasmid DNA containing sequence derived from a Chinese genotype III/2a isolate of hepatitis C virus

OBJECTIVE To induce antibody response against E2 glycoprotein derived from a Chinese genotype III/2a isolate of hepatitis C virus (HCV) in BALB/C mice by plasmid DNA-based immunization.

METHODS Plasmid p3W14 containing E2/NS1 gene derived from a Chinese genotype III/2a isolate of HCV, which was verified to express 70 kDa E2 glycoprotein in NIH3T3 cells, was used for direct intramuscular injection in BALB/C mice. Specific antibody to E2 glycoprotein was detected by recombinant E2 protein.

RESULTS Specific anti-E2 antibody could be detected in mice inoculated with p3W14(5/6). Titer of antibody ranged from 1:15 to 1:120.

CONCLUSIONS Successfully inducing anti-E2 antibody in mice by plasmid DNA-based immunization containing E2/NS1 gene from a Chinese genotype III/2a isolate of HCV is the first time in China.

     

乙型肝炎病毒x基因转基因小鼠模型的建立及培育

目的：建立可表达乙型肝炎病毒X蛋白的HBx基因（adr亚型）转基因小鼠模型，以研究x基因的功能。方法：采用基因重组技术构建含有CMV启动子和HBx基因（adr亚型）开放阅读框（ORF）的真核表达载体pcDNA3-HBx。该载体经Sal I限制性内切酶线性化后，琼脂糖电泳回收目的的片段，用原核显微注射法将其注射入雄原核，制备转基因小鼠。复合PCR法在基因组水平筛选HBx基因转基因小鼠founder；免疫组织化学法在蛋白水平检测X蛋白在这些小鼠中的表达情况。结果：成功构建了HBx基因真核表达载体pcDNA3-HBx。经原核显微注射法将目的片段注射入受精卵雄原核后，出生并存活了11只新生鼠，经PCR检测后获得5只founder转基因小鼠，命名为C57－TgN(HBx)SMMU。免疫组织化学检测发现5只PCR阳性小鼠的肝细胞质中均有X蛋白的表达。将这5只转基因小鼠与正常同系异性小鼠交配，进行传代培育，复合PCR法筛选阳性转基因小鼠，目前已传至F4代。结论：本研究成功地产生了稳定遗传HBx基因并表达X蛋白的转基因小鼠C57－TgN（HBx)SMMU，它将有利于体内研究HBx基因在肝细胞癌发生中的作用。     

HBV感染研究中Th细胞分化的调节和意义

Th细胞免疫失衡是乙型肝炎病毒(HBV)慢性感染的重要机制之一。Th细胞分化受细胞因子及其受体、T细胞受体(TCR)-抗原提呈细胞(APC)-抗原/主要组织相容性复合物、细胞信号转导、HBV基因型及变异、调节性T细胞(Treg)等方面的影响。了解Th细胞分化情况,对HBV感染的防治有重要指导意义。一是有助于了解乙型肝炎疫苗的接种效果和改进疫苗;二是指导研发抗病毒药物和治疗性疫苗,通过上调Th1免疫反应或调节Th2优势反应向Th1优势反应转化来治疗慢性乙型肝炎。     

最低限度免疫定义基因表达的HCV多表位DNA疫苗对小鼠细胞免疫的诱导作用

目的：探讨采用最低限度免疫定义基因表达法制备丙型肝炎病毒（HCV）多表位DNA疫苗的可行性,阐明该方法在疫苗领域可能的应用价值。方法：采用人工合成和PCR方法制备长度为1 346 bp的含有CMV启动子、HCV 1b亚型多表位和牛生长激素（BCG）多聚腺苷酸序列的最低限度免疫定义基因表达DNA疫苗,命名为M-HCV-epi;同时制备结构基因被非HCV同源的DNA序列替换的相同长度DNA片段作为对照,命名为V-pcDNA3.1。12只ICQ小鼠随机分为实验组（n=6）和对照组（n=6）,分别采用M-HCV-epi DNA和V-pcDNA3.1 DNA各20 μg皮下注射。QRT-PCR法检测免疫后小鼠脾细胞中干扰素γ（IFN-γ）mRNA的表达水平;人工合成3个HCV 1b亚型表位多肽和1条对照多肽。用上述合成的多肽刺激免疫后小鼠脾细胞,ELISA法检测脾细胞刺激上清中IFN-γ水平。结果：与对照组比较,实验组小鼠脾细胞中IFN-γ mRNA表达水平升高（1.50±0.18）倍（P<0.05）;加入aa35-44多肽的实验组小鼠脾细胞培养上清中IFN-γ水平较对照组明显升高（P<0.05）。结论：最低限度免疫定义基因表达法制备HCV多表位DNA疫苗可诱导细胞免疫反应,该方法在DNA疫苗领域可能具有应用价值。