新型乙型肝炎病毒表面抗原中蛋白核酸疫苗的免疫原性研究

目的 观察新型乙型肝炎病毒（HBV）表面抗原中蛋白（MHBs)核酸疫苗的免疫原性。方法 以新型人体应用载体质粒pSW3891构建MHBs核酸疫苗（pSW3891/MHBs/adr)，对照组和实验组Balb/c小鼠分别基因枪法免疫对照载体质粒（pSW3891)和MHBs核酸疫苗，采用酶联免疫吸附试验检测抗HBs,LDH释放测定法检测小鼠特异性细胞毒T淋巴细胞（CTL）杀伤活性。结果 MHBs核酸疫苗可在体外高效表达，免疫小鼠后可产生高滴度抗HBs，血清阳性终点滴度达1：97200,小鼠脾细胞HBs特异性细胞毒性T淋巴细胞（CTL）杀伤活性达78.81%。结论 新型MHBs核酸疫苗在Balb/c小鼠实验中表现出良好的体液和细胞免疫原性。     

血清HBsAg与HBVDNA定量关系分析

目的定量检测血清乙型肝炎病毒表面抗原（HBsAg）浓度与乙型肝炎病毒DNA（HBVDNA）,并探讨两者的相关关系及临床应用价值。方法随机选取HBsAg阳性的患者135例,采用化学发光法（CLIA）定量检测其血清中HBsAg、乙型肝炎病毒e抗原（HBeAg）,同时采用实时荧光定量聚合酶链反应（FQ-PCR）检测其血清中HBVDNA载量。结果 135例患者中HBeAg阳性的61例,HBVDNA阳性的87例,其HBsAg和HBeAg浓度值与HBVDNA载量的相关系数（r）分别为：0.583（P〈0.01）、0.550（P〈0.01）,HBsAg与HBeAg的r值为：0.699（P〈0.01）。结论血清HBsAg浓度与HBVDNA载量及HBeAg浓度均成高度正相关,可较好的反应HBV的复制水平,便于临床开展应用。     

Preparation and immunological effectiveness of a Swine influenza DNA vaccine encapsulated in PLGA microspheres

Optimal preparation conditions of DNA vaccine against swine influenza encapsulated in Poly (D,L)-lactic-co-glycolic acid (PLGA) microspheres were determined. The microspheres were prepared by an emulsion-evaporation method using PLGA as the biodegradable matrix forming polymer. Using the optimal preparation conditions, PLGA microspheres containing the DNA vaccine were produced with good morphology as evident from scanning electron micrographs, high encapsulation rate and high stability. The transfection test indicated that the vaccine could be expressed as an antigen in cells and maintained good bioactivity. Moreover, these results demonstrated that the PLGA microspheres containing DNA vaccine can be used to achieve prolonged release of plasmid DNA. These results have laid a foundation for further development before ultimate industrial application.     

乙型肝炎表面抗原定量与原发性肝癌的相关性研究

目的 比较慢性乙型肝炎(CHB)与HBV相关性原发性肝癌患者中乙肝表面抗原(HBsAg)和HBV DNA 载量情况。方法 采用化学发光法检测403例CHB及肝细胞肝癌(HCC)患者血清乙型肝炎病毒标志物滴度,采用荧光定量PCR技术检测患者血清HBV DNA载量。403例患者按照临床诊断分为CHB组(209例)和HCC组(194例)。结果 CHB组:血清HBsAg 滴度≥250 IU/mL 者占89.00%; HBV DNA≥1 000 copies /mL 者占88.40%。HCC 组: 血清HBsAg 滴度≥250 IU/mL 者占79.90%; HBV DNA≥1 000 copies /mL 者占67.70%。两组HBsAg≥250 IU/mL患者的比例差异、HBV DNA≥1 000 copies/mL 患者的比例差异均有统计学意义(P<0.05)。结论 与CHB相比,HBV相关性原发性肝癌患者HBsAg、HBV DNA载量较低。     

表面抗原定量在慢性乙型肝炎抗病毒治疗中的研究现状

慢性乙型肝炎在我国仍是一大疾病,其疾病进展及并发症严重威胁到我国国民健康,随着抗病毒治疗的普及,疾病的进程得到一定的延缓甚至逆转,表面抗原定量在慢性乙型肝炎病人的抗病毒治疗中可起到指导作用,该研究就目前表面抗原定量在慢性乙型肝炎治疗中的研究进展进行简单分析。     

新生乙型肝炎疫苗接种者无应答调查分析

目的通过新生入学体检测HBsAg和HBs-Ab,了解学生携带乙型肝炎病毒的状态和乙型肝炎疫苗接种者无应答情况。方法用酶联免疫吸附试验(ELISA)测定入学新生人群的HBsAg和HBs-Ab。结果体检总人数为1178人,其中HBsAg阳性170人,占14.43%,HBs-Ab阴性人数为246,占20.88%,经调查接种后抗体阴性者仍然有144人,占16.04%。结论通过调查了解新生中HBsAg携带者和接种乙肝疫苗后无应答者,有助于学生和学校采取必要的措施,尽可能减少乙型肝炎病毒在校园内传播,对乙型肝炎疫苗接种无应答者提出合理的建议,让学生了解自己的免疫状态,以便采取进一步的补救措施。     

乙型肝炎疫苗应对全球乙型肝炎病毒基因多态性的有效性

基因重组乙型肝炎(乙肝)疫苗是预防乙肝的安全有效方法.乙肝病毒(HBV)具有基因多态性,目前已知共有10个基因型,全球所用的乙肝疫苗为A2基因型重组乙肝疫苗.临床前实验和临床试验结果表明,HBV A2疫苗对非A2基因型感染具有交叉防御作用,能够有效预防所有已知HBV基因型所造成的感染和临床疾病.     

Modulation of immune response induced by co-administration of DNA vaccine encoding HBV surface antigen and HCV envelope antigen in BALB/c mice

Plasmid DNA vaccines encoding the hepatitis B virus (HBV) surface and hepatitis C virus (HCV) envelope antigens, respectively, were constructed, and attempt were made to find the possibility of a divalent vaccine against HBV and HCV. The expression of each plasmid in Cos-1 cells was confirmed using immunocytochemistry. To measure the induced immune response by these plasmids in vivo, female BALB/c mice were immunized intramuscularly with 100 microg of either both or just one of the plasmids. Anti-HBV and HCV-specific antibodies and related cytokines were evaluated to investigate the generation of both humoral and cellular immune responses. As a result, specific anti-HBV and anti-HCV serum antibodies from mice immunized with these plasmids were observed using immunoblot. The levels of IL-2 and RANTES showing a Th1 immune response were significantly increased, but there was no change in the level of IL-4 (Th2 immune response) in any of the immunized groups. Compared with each plasmid DNA vaccine, the combined vaccine elicited similar immune responses in both humoral and cell-mediated immunities. These results suggest that the combined DNA vaccine can induce not only comparable immunity experimentally without antigenic interference, but also humoral and Th1 dominant cellular immune responses. Therefore, they could serve as candidates for a simultaneous bivalent vaccine against HBV and HCV infections.     

无症状乙型肝炎表面抗原阳性患者206例丙氨酸转氨酶异常临床分析

目的了解无症状乙型肝炎表面抗原(HBsAg)阳性者丙氨酸转氨酶(ALT)及乙型肝炎病毒复制的情况。方法对206例无症状HBsAg阳性者进行ALT、乙型肝炎五项及乙型肝炎病毒脱氧核糖核酸(HBV-DNA)的检测。结果无症状HBsAg阳性者中有19.9%的患者存在ALT的异常,但造成ALT异常的因素与性别差异无关;在ALT异常的无症状HBsAg阳性者中HBV-DNA的阳性率为68.2%。结论对于临床无症状的HBsAg阳性人群应定期检查,做到早发现、早治疗,以延缓疾病进展,减少肝硬化、原发性肝癌的发生。     

乙肝病毒载量测定与前S1抗原、谷氨酸氨基转移酶测定之比较及其临床价值

目的研究乙肝患者血清中乙肝病毒的核酸载量与乙肝病毒前S1抗原阳性以及谷氨酸氨基转移酶(ALT)水平之间的相关性与符合率,以及三者检测的临床价值。方法用荧光定量聚合酶链反应的方法检测334份怀疑HBV感染的血清中HBV—DNA的病毒拷贝数．同时检测血清中ALT和乙肝前Sl抗原。结果275份HBV—DNA拷贝数大于10^3copies／ml样本中,前S1阳性率80．0％(220／275)。ALT异常率为69．5％(191／275)：而59份HBV—DNA拷贝数小于10^3copies／ml的血清．其前S1抗原阳性率为25．4％．ALT的异常率为22％．且前S1抗原的阳性率和ALT异常率随着HBV—DNA载量升高而增加。结论HBV—DNA的载量与前S1抗原的阳性、ALT水平三者之间,有符合性和互补性,因此三者联合检测．对全面了解HBV感染者的病情、以及药物治疗的评价和预后判断等均有较高的临床应用价值。     

抗变异乙型肝炎表面抗原单克隆抗体与8种乙型肝炎检测试剂盒检测效果的比较

目的评价本实验室制备的抗变异乙型肝炎(乙肝)表面抗原单克隆抗体检测野生及变异表面抗原的能力。方法用酶联免疫吸附试验(ELISA)评价本实验室制备的抗变异乙肝表面抗原的单克隆抗体,以及国内常用的8种乙肝检测试剂盒,对3种野生乙肝表面抗原和15种变异乙肝表面抗原的检测效果。结果本实验室制备的抗乙肝变异表面抗原单克隆抗体对变异表面抗原的检测能力明显优于国内常用检测试剂盒。结论该单克隆抗体为进一步研制高敏感性乙肝检测试剂盒提供了前提。     

微卡联合乙肝疫苗、双嘧达莫治疗乙肝病毒携带者临床观察

目的 探讨乙型肝炎病毒携带者的治疗方法。方法 治疗组26例,应用微卡＋乙肝疫苗＋双嘧达莫;对照组25例不用药物。对慢性无症状乙型肝炎病毒携带者治疗观察18个月。结果 治疗结束后,综合疗效分析,治疗组：显效11.54%,有效69.23%,无效19.23%,总有效率80.77%。对照组：显效4%,有效12.00%,无效84.00%,总有效率16.00%。经统计学处理,治疗组与对照组比较差异有统计学意义（χ2=21.3948,P〈0.01）。结论 微卡联合乙肝疫苗和双嘧达莫治疗AsC具有较好的抑制病毒复制的作用。     

Neutralization of hepatitis B virus (HBV) by human monoclonal antibody against HBV surface antigen (HBsAg) in chimpanzees

The virus neutralizing efficacy of HB-C7A, a human monoclonal antibody raised against the surface antigen of hepatitis B virus (HBsAg), was proved using hepatitis B virus (HBV)-na?ve chimpanzees. One control chimpanzee which received 100CID(50) of HBV, subtype adw, without HB-C7A antibody became infected by HBV as evidenced by the appearance of HBV DNA on week 10 and subsequent appearance of HBsAg, anti-HBc and anti-HBs in the serum. Two experimental chimpanzees were inoculated intravenously with same dose of HBV as the control chimpanzee, which was previously incubated with 0.1mg and 10mg of HB-C7A antibody prior to inoculation. HBV infection was not observed in the antibody-treated chimpanzees during 12 months of follow-up, exhibiting neither detectable HBsAg nor anti-HBc antibody. This work demonstrates the neutralization of HBV by HB-C7A monoclonal antibody and shows the possibility of prevention of HBV infection using this antibody in liver transplantation and exposure to HBV.     

近年健康体检者乙型肝炎表面抗原携带情况结果分析

目的：了解健康体检者乙型肝炎表面抗原（HBsAg）携带情况,探讨更加有效的预防控制措施。方法：对2007年9月～2010年8月的37970名来该院进行健康体检者的静脉血样,采用酶联免疫吸附试验法（ELISA）检测HBsAg。结果：37970名健康体检者中,HBsAg阳性1416名,阳性率为3.7%。其中男性阳性率为4.0%,女性阳性率为3.3%。结论：2007年9月～2010年8月乙型肝炎病毒携带者呈逐年上升趋势,并且男性高于女性,HBsAg阳性者集中于46～60岁年龄组,做好HBsAg监测工作,预防和降低乙型肝炎感染刻不容缓。     

乙肝疫苗联合免疫球蛋白在阻断乙肝母婴传播中的临床疗效观察

目的探讨分析乙肝疫苗联合免疫球蛋白在乙肝母婴传播途径阻断中的作用效果。方法选取从2010年1月至2012年11月在我院进行产前检查的住院分娩的200例HBsAg阳性孕妇及脐血为研究对象,所有孕妇的肝功能均正常。按照平均随机抽样的原则平均分为对照组和干预组,各为100例孕妇。对照组的新生儿在出生后24 h内接种乙肝疫苗联合免疫球蛋白100IU;干预组的孕妇在孕周为28、32和36周时,分别注射200IU的免疫球蛋白各1次,其新生儿在出生后的处理与对照组相同。所有病例均进行成功随访,观察接种后12个月两组新生儿乙肝五项检查情况。结果干预组和对照组的12个月新生儿HBsAg和HBsAb阳性率分别为1%、94%和6%、75%,两组间的差异均有有统计学意义(P<0.05)。结论对新生儿肌内注射免疫球蛋白联合乙肝疫苗接种对乙肝母婴阻断中的作用效果显著,尤其是在分娩前进行免疫球蛋白产前干预具有良好的免疫阻断作用,降低了新生儿感染乙肝的概率安全有效,值得临床推广应用。     

Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses

This study investigates the efficacy of surface-modified microspheres of hepatitis B surface antigen (HBsAg) in eliciting systemic and mucosal immune responses. Positively charged poly(d,l-lactic-co-glycolic acid) microspheres were prepared by a double-emulsion solvent-evaporation method with cationic agents—stearylamine and polyethylenimine—in the external aqueous phase. Formulations were characterized for morphology, size, density, aerodynamic diameter, entrapment efficiency and in vitro drug-release profile. Immunization was performed after pulmonary administration of the formulations to female Sprague–Dawley rats and the immune response was monitored by measuring IgG levels in serum and secretory (sIgA) levels in salivary, vaginal and bronchoalveolar lavage fluids. The cell-mediated immune response was studied by measuring cytokine levels in spleen homogenates, and a cytotoxicity study was performed with Calu-3 cell line. The aerodynamic diameter of the particles was within the respirable range, with the exception of stearylamine-modified particles. Zeta potential values moved from negative (−6.76 mV) for unmodified formulations to positive (+0.515 mV) for polyethylenimine-modified particles. Compared to unmodified formulations, polyethylenimine-based formulations showed continuous release of antigen over a period of 28–42 days and increased levels of IgG in serum and sIgA in salivary, vaginal and bronchoalveolar lavage. Further, cytokine levels—interferon γ and interleukin-2—were increased in spleen homogenates. The viability of Calu-3 cells was not adversely affected by the microparticles. In summation, this study establishes that positive surface charges on poly(d,l-lactic-co-glycolic acid) particles containing HBsAg enhances both the systemic and mucosal immune response upon immunization via the respiratory route.     

某校大学新生乙肝疫苗接种情况及其影响因素的调查分析

目的：为促进大学生的乙肝防治工作提供参考。方法：采用问卷调查法,对大学新生中乙肝“两对半”全阴的学生进行乙肝疫苗接种情况调查,并分析其影响因素。结果：共调查3647人,乙肝疫苗接种率为85．66％（3124／3647）,接种人群中全程接种率为96．38％（3011／3124）。家庭人均月收入越高、父母文化程度越高、父母就职于企事业单位和医疗系统、周围有乙肝患者、对乙肝传播途径认识正确者、对乙肝疫苗信任者、对乙肝危害认识正确者更易接种乙肝疫苗,且接种针次更多。结论：家庭收入、父母文化程度、父母职业、对乙肝传播途径的认知、对乙肝疫苗的信任、对乙肝危害的认识是大学新生乙肝疫苗接种行为的主要影响因素。学校应加大对大学新生的乙肝防治宣传工作,以提高整个校园及周边的接种率。     

脂质体VEGFR2胞外区基因肿瘤疫苗的制备及其免疫激活体外抗肿瘤活性研究

目的:制备脂质体(LP)携带VEGFR2胞外区(exVEGFR2)肿瘤抗原pCMV质粒复合物基因疫苗,为肿瘤免疫治疗提供新的主动免疫疗法。方法:RT-PCR扩增含2个KpnⅠ和XbaⅠ限制性酶切位点的exVEGFR2序列,将其与pCMV质粒连接,构建pCMV/exVEGFR2质粒。Western blot检测exVEGFR2体外表达水平。用制备好的脂质体pCMV/exVEGFR2复合物(LP-pCMV/exVEGFR2)免疫C57BL/6小鼠,ELISA法检测其免疫激活活性。利用51 Cr释放实验分析CTLs介导的细胞毒活性。结果:成功扩增到exVEGFR2序列,并构建pCMV/exVEGFR2质粒;只有在pCMV/exVEGFR2转染COS-7细胞中,检测到与目的蛋白序列大小一致的相对分子质量为90000的VEGFR2特异性条带。LP-pCMV/exVEGFR2免疫小鼠6周后,ELISA法检测到强烈的特异性抗VEGFR2免疫激活效应;其免疫T细胞能够有效地介导体外VEGFR2阳性的CT-26结肠癌细胞毒性。结论:LP-pCMV/exVEGFR2能够有效地激活小鼠产生特异性抗VEGFR2免疫应答效应,并产生体外抗肿瘤细胞免疫毒性,为后期研究主动免疫治疗VEGFR2阳性肿瘤奠定实验基础。     

Comparison of in vivo fate and immunogenicity of hepatitis B surface antigen incorporated in cationic and neutral liposomes

To compare cationic liposomes (CatL) and neutral liposomes (NeuL), as a vaccine carrier, the in vivo fate and immunogenicity of hepatitis B surface antigen (HBsAg), incorporated in CatL and NeuL, were investigated. CatL, composed of phosphatidyl choline (PC) and stearyl amine (SA) with a molar ratio of 9:1, showed a 2.5-fold higher incorporating efficiency of HBsAg than NeuL composed of PC alone. Most of HBsAg incorporated in both liposomes existed in an antibody-available form on the outer surface of liposomes. After intramuscular injection to rats, HBsAg in CatL resided at the injection site for a longer period than that in NeuL with terminal half lives of 52.5 and 42.9h, respectively. However, HBsAg in NeuL was more efficiently taken up by the lymphatic organs and spleen than that in CatL. Furthermore, the group treated with HBsAg in NeuL showed earlier sero-conversion with higher anti-HBsAg titre than the group treated with HBsAg in CatL. Sero-conversion rates (SCRs) in both CatL- and NeuL-treated animals were 100% after every injection carried out, except the primary injection of CatL. These results demonstrate that CatL can enhance the retention of incorporated antigen at the injection site, compared with NeuL. However, the production of antibody by HBsAg in NeuL is more effective than that by HBsAg in CatL, probably due tothe higher lymphatic targeting ability of NeuL. Thus, NeuL appears to be a better carrier for HBsAg than CatL.     

Enhancement of immune response of HBsAg loaded poly (L-lactic acid) microspheres against Hepatitis B through incorporation of alum and chitosan

Purpose: Poly (L-lactic acid) (PLA) microparticles encapsulating Hepatitis B surface antigen (HBsAg) with alum and chitosan were investigated for their potential as a vaccine delivery system.

Methods: The microparticles, prepared using a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method with polyvinyl alcohol (PVA) or chitosan as the external phase stabilising agent showed a significant increase in the encapsulation efficiency of the antigen.

Results: PLA-Alum and PLA-chitosan microparticles induced HBsAg serum specific IgG antibody responses significantly higher than PLA only microparticles and free antigen following subcutaneous administration. Chitosan not only imparted a positive charge to the surface of the microparticles but was also able to increase the serum specific IgG antibody responses significantly.

Conclusions: The cytokine assays showed that the serum IgG antibody response induced is different according to the formulation, indicated by the differential levels of interleukin 4 (IL-4), interleukin 6 (IL-6) and interferon gamma (IFN-γ). The microparticles eliciting the highest IgG antibody response did not necessarily elicit the highest levels of the cytokines IL-4, IL-6 and IFN-γ.