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1.
Objective To develop an effective and broad immune protective H5N1 vaccine.Methods We first developed two recombinant vaccinia ( Tiantan strain) virus ( rTTV ) based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin(HA) and matrix protein 2(M2), or bicistron expressing the neuraminidase(NA) and matrix protein 1 (M1). The expression of H5N1 protein in rTTVs was confirmed. We immunized the BALB/c mice twice with two kind of dose ( 104 PFU, 107 PFU)using different combination. Subsequently, we assessed the humoral and cellular immune response in vaccinated mice. Results Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HA- and NAspecific antibody level and IFN-γ secreting form cell(SFC) with either single dose of 107 PFU or twice dose of 104 PFU or 107 PFU. We also detected significant neutralizing antibody and matrix-specific immune response. In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level of M2-specific antibody than that with single of rTTV-based H5N1 vaccine. Conclusion rTTVbased H5N1 vaccines in this study elicited board array of immunity and our study offers a promising alternative H5N1 vaccine candidates with favorable potential to prevent various H5N1 pandemic.  相似文献   

2.
Objective To evaluate the seasonal influenza spilt vaccine's immunogenicity and the 50% effective dose ( ED50a ) of hemagglutin ( HA ) that can make 50% of the mice hemagglutination inhibition antibody ( HI ) titers to 40. Methods The 2008-2009 seasonal influenza spilt vaccine's two components, with HA from H1N1 and H3N2 influenza virus respectively, were used as a model. Mice were immunized once or twice with different doses, and the HI antibody titers were tested to determine the immunization procedure and to evaluate the immugenicity of seasonal influenza spilt vaccine in mice;Consequently, HI antibody response kinetics of the two components were observed to determine the time point when the HI antibody titer reached the peak point; Finally, mice were immunized with different doses of HA to evaluate the ED50a that can make 50% of mice HI titers reach 40. Results Immunization procedures study showed that one-dose of seasonal influenza vaccine induced the HI antibody titem ranged from 10 to 120, while two-dose of influenza vaccine improved the HI antibody titer 10-100 times as compared with one dose; antibody kinetics study suggested that the time point of HI antibody produced to peak is 28-35 days post one dose immunization ; and the ED50a detection results indicated that one dose of 1.5 μg HA could make 50% of the mice HI antibody titer reach 40. Conclusion Seasonal influenza spilt vaccine is very immunogenic in mouse; the time point of HI antibody produced to peak is 28-35 days post one dose immunization; and the ED50a of HA is 1.5 μg, which can make 50% of the mice HI titer reach 40. The experimental results provided foundation for the establishment of influenza vaccine evaluation system based on seasonal influenza vaccine.  相似文献   

3.
目的 观察柯萨奇病毒B3(Coxsackievirus B3,CVB3)衣壳蛋白VP1、表达VP1蛋白的重组腺病毒rAd/VP1和重组质粒pcDNA3/VP1的免疫效果.方法 用原核细胞表达VP1蛋白并纯化、扩增重组腺病毒rAd/VP1,扩增并提取真核表达质粒pcDNA3/VP1.BALB/c小鼠随机分为4组,每组18只,分别在股四头肌注射VP1蛋白、rAd/VP1、pcDNA3/VP1和PBS.VP1蛋白组和pcDNA3/VP1组免疫3次,间隔3周;rAd/VP1组免疫2次,间隔2周.VP1蛋白、pcDNA3/VP1和rAd/VP1每次每只注射剂量分别为50μg、100μg和1.2×107PFU.用ELISA法和微量中和试验法检测各次免疫后血清CVB3特异性IgG抗体和中和抗体滴度;末次免疫后3周,CCK-8法检测脾脏淋巴细胞的CTL杀伤活性;用致死量CVB3攻击小鼠后,检测血中病毒滴度并观察动物的存活情况.结果 VP1蛋白组血清特异性IgG抗体和中和抗体滴度明显高于其他实验组(P<0.05),而脾脏淋巴细胞CTL杀伤活性低于rAd/VP1组(P<0.05);致死量病毒攻击后,VP1蛋白组血中病毒滴度低于pcDNA3/VP1和rAd/VP1组(P<0.05),生存率明显高于这两组(P<0.05).结论 VP1蛋白疫苗能诱导较高水平的体液免疫应答,对动物有明显的免疫保护作用,免疫效果优于质粒pcDNA3/VP1和重组腺病毒rAd/VP1.
Abstract:
Objective To compare the immune effects of Coxsackievirus B3 (CVB3) capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte(CTL) killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose( CCID50 ) assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups( P <0.05 ). While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group( P <0. 05). Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups ( P < 0.05 ), while survival rate was significantly higher than these two groups ( P < 0.05 ).Conclusion VP1 protein induced higher level of humoral immune response and acquired obvious immune protection effects in mice. The immunizing potency of VP1 protein vaccine surpassed plasmid pcDNA3/VP1or recombinant adenovirus rAd/VP1. It appeared to be a promising candidate among the three different vaccines.  相似文献   

4.
Objective To investigate the adjuvant effect of dimo-thylidioctyl ammonium bromide (DDA) and/or DDA-BCG polysaccharide nucleic acid( BCG-PSN), which was combined with a Mycobacterium tuberculosis fusion protein AMM ( Ag 8 5 B - MPT64190-198 - Mtb8.4 ) to boost BCG primed immunization. Methods DDA with or without BCG PSN was mixed with the fusion protein AMM to construct the boosting vaccine. Mice were immunized with BCG and then boosted twice with AMM formulated with the adjuvant DDA with or without BCG-PSN. PBS or BCG vaccination without boosting was used as control. The humoral and cell-mediated immune responses were analyzed by ELISA and ELISPOT. Moreover, the protective efficacy of BCG prime-AMM subunit vaccine boosting against Mycobacterium tuberculosis infection was analyzed. Results With in vitro stimulation of Ag85B and PPD( purified protein derivative) antigen, the number of IFN-γ secreting cells from the mice boosted twice by AMM/DDA/BCG-PSN and AMM/DDA were higher than BCG and PBS group (P <0.05). The CFU in lungs of mice boosted with AMM/DDA/BCG-PSN was less than that of PBS group(P <0.05), while the CFU of AMM/DDA-boosted mice was less than that of BCG and PBS group(P < 0.05).However, fewer lesions were seen in lungs of mice immunized with BCG alone or BCG-prime-AMM/DDA/BCG-PSN boosting than the other groups. Conclusion DDA is an idea adjuvant for tuberculosis subunit vaccine;BCG-PSN might play a role in alleviating the immunity-mediated pathology.  相似文献   

5.
Objective To investigate the adjuvant effect of dimo-thylidioctyl ammonium bromide (DDA) and/or DDA-BCG polysaccharide nucleic acid( BCG-PSN), which was combined with a Mycobacterium tuberculosis fusion protein AMM ( Ag 8 5 B - MPT64190-198 - Mtb8.4 ) to boost BCG primed immunization. Methods DDA with or without BCG PSN was mixed with the fusion protein AMM to construct the boosting vaccine. Mice were immunized with BCG and then boosted twice with AMM formulated with the adjuvant DDA with or without BCG-PSN. PBS or BCG vaccination without boosting was used as control. The humoral and cell-mediated immune responses were analyzed by ELISA and ELISPOT. Moreover, the protective efficacy of BCG prime-AMM subunit vaccine boosting against Mycobacterium tuberculosis infection was analyzed. Results With in vitro stimulation of Ag85B and PPD( purified protein derivative) antigen, the number of IFN-γ secreting cells from the mice boosted twice by AMM/DDA/BCG-PSN and AMM/DDA were higher than BCG and PBS group (P <0.05). The CFU in lungs of mice boosted with AMM/DDA/BCG-PSN was less than that of PBS group(P <0.05), while the CFU of AMM/DDA-boosted mice was less than that of BCG and PBS group(P < 0.05).However, fewer lesions were seen in lungs of mice immunized with BCG alone or BCG-prime-AMM/DDA/BCG-PSN boosting than the other groups. Conclusion DDA is an idea adjuvant for tuberculosis subunit vaccine;BCG-PSN might play a role in alleviating the immunity-mediated pathology.  相似文献   

6.
Lack of an appropriate small animal model remains a major hurdle for studying the immunotolerance and immunopathogenesis induced by hepatitis B virus (HBV) infection. In this study, we report a mouse model with sustained HBV viremia after infection with a recombinant adeno-associated virus (AAV) carrying a replicable HBV genome (AAV/ HBV). Similar to the clinical HBV carriers, the mice infected with AAV/H BV were sero-negative for antibodies against HBV surface antigen (HBsAg). Immunization with the conventional HBV vaccine in the presence of aluminum adjuvant failed to elicit an immune response against HBV in these mice. To identify a vaccine that can potentially circumvent this tolerance, the TLR9 agonist CpG was added to HBsAg as an adjuvant. Vaccination of mice with HBsAg/CpG induced not only clearance of viremia, but also strong antibody production and T-cell responses. Furthermore, both the DNA replication and protein expression of HBV were significantly reduced in the livers of AAV/H BV-infected mice. Accordingly, AAV/HBV-infected mice may be used as a robust model for investigating the underlying mechanism(s) of HBV immunotolerance and for developing novel immunotherapies to eradicate HBV infections.  相似文献   

7.
There are no approved flaviviral therapies and the development of vaccines against flaviruses has the potential of being undermined by antibody-dependent enhancement(ADE).The flavivirus nonstructural protein 1(NS1)is a promising vaccine antigen with low ADE risk but has yet to be explored as a broad-spectrum therapeutic antibody target.Here,we provide the structural basis of NS1 antibody cross-reactivity through cocrystallization of the antibody 1G5.3 with NS1 proteins from dengue and Zika viruses.  相似文献   

8.
Exosomes (EXO) derived from dendritic cells (DC) and tumor cells have been used to stimulate antitumor immune responses in animal models and in clinical trials. However, there has been no side-by-side comparison of the stimulatory efficiency of the antitumor immune responses induced by these two commonly used EXO vaccines. In this study, we selected to study the phenotype characteristics of EXO derived from a transfected EG7 tumor cells expressing ovalbumin (OVA) and OVA-pulsed DC by flow cytometry. We compared the stimulatory effect in induction of OVA-specific immune responses between these two types of EXO. We found that OVA protein-pulsed DCovA-derived EXO (EXODC) can more efficiently stimulate naive OVA-specific CD8+ T cell proliferation and differentiation into cytotoxic T lymphocytes in vivo, and induce more efficient antitumor immunity than EG7 tumor cell-derived EXO (EXOEG7). In addition, we elucidated the important role of the host DC in EXO vaccines that the stimulatory effect of EXO is delivered to T cell responses by the host DC. Therefore, DC-derived EXO may represent a more effective EXO-based vaccine in induction of antitumor immunity.  相似文献   

9.
Objective To investigate the effects of in vivo electroporation on plasmid mediated reporter gene expression and immunogenicity of DNA vaccine. Methods Luciferase expression plasmid was administered intramuscularly to BALB/c mice at 8μg and 40μg dosage level through injection with or without eletroporation Luciferase expression level in murine muscle was detected by IVIS imaging system 24 h after injection. DNA vaccine plasmid p1.0-gp1455m carrying codon-optimized env gene of CN54 strain ( HIV-1 CRF07_BC) was administered to mice at dosages of 8μg and 40μg through the two approaches mentioned above. Mice were immunized at week 0,2 and 4. Env-specific immune responses were detected at two weeks post the second and the third vaccinations. Env-specific antibody immune responses were determined by ELISA. Euv-specific cellular immune responses were determined by IFN-γ ELISPOT. Results Luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. Results of ELISA and ELISPOT revealed that in vivo eletroporation could significantly enhance both the humoral and cellular immune responses induced by DNA vaccination. The responses induced by electrodelivered p1.0-gp1455m at 8 μg dosage were better than those induced by simple intramuscular injection with 40 μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation. Conclusion In vivo electroporation was capable of enhancing both the plasmid-mediated gene expression and immunogenicity of DNA vaccine.  相似文献   

10.
The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod.gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod.gag, rAd5-mod.gag or DNA and were boosted after 3 weeks. To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIV-specific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.  相似文献   

11.
目的 研发高效广谱的人高致病性禽流感病毒H5N1实验疫苗.方法 首先构建了含H5N1(安徽株)结构基因[血凝素(HA)、神经氨酸酶(NA)、基质蛋白M1与M2]的两个双顺反子(HAop/M2,NAop/M1)重组痘苗病毒(rTTV天坛株)疫苗,采用不同剂量(104 PFU或107PFU)或组合(疫苗单独或联合)方式于0、4周二次免疫BALB/c小鼠,初步比较分析抗原特异的体液(HA血凝抑制抗体、NA特异性抗体、中和抗体)与细胞免疫应答(IFN-γ ELISPOT)特点.结果 重组痘苗病毒疫苗可有效表达H5N1靶抗原;高剂量组的重组痘苗病毒疫苗可快速激发较强的针对各个抗原的抗体与针对血凝素与神经氨酸酶蛋白的细胞免疫应答,含血凝素蛋白的重组痘苗病毒疫苗亦可诱导明显的中和抗体;但各组重组痘苗病毒疫苗所激发的针对基质蛋白(M1,M2)的细胞免疫应答均较弱;两个双顺反子(HAop/M2,NAop/M1)重组痘苗病毒疫苗联合应用所激发的针对基质蛋白2(M2)的体液免疫应答明显强于单双顺反子(HAop/M2)疫苗单独应用.结论 本研究中制备的各组重组痘苗病毒疫苗可诱导多个抗原特异的体液与细胞免疫应答,该研究为新型H5N1疫苗的研发及免疫方案的优化奠定了基础.  相似文献   

12.
目的:预测H5N1亚型禽流感病毒血凝素Th和B细胞相关抗原表位,并初步分析其抗原性.方法:依据近年H5N1亚型禽流感病毒流行趋势,下载得到相关HA蛋白氨基酸序列.进行生物信息学综合分析预测,获得Th和B细胞相关抗原表位,并比较其保守性和特异性.通过BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清,初步鉴定候选表位抗原性.结果:综合多项预测及空间构象模拟结果,我们获得了三条候选Th和B细胞表位,分别为HA141~155、HA206~223、HA302~316.候选表位处于H5N1亚型禽流感HA1 蛋白序列上相对保守的区域内,且与目前流行的H5N1亚型禽流感病毒HA相应区域具有较好的一致性.而不同候选表位在BALB/c小鼠和SPF鸡H5N1亚型禽流感病毒阳性血清反应中显示了不同抗体结合能力,预示了其成为功能表位的可能.结论:所筛选的表位具有成为H5N1亚型禽流感病毒HA Th和B细胞相关抗原表位的可能.本研究为深入揭示流感病毒感染与免疫机制,H5N1亚型禽流感功能表位认知及表位疫苗研究奠定了基础.  相似文献   

13.
水禽类被认为是甲型流感病毒的自然基因储存库,为人和低等动物各亚型流感病毒的来源.流感病毒唾液酸受体结合特异性在流感病毒种属范围限制中起重要作用.一般认为,水禽类仅表达SA2-3Gal受体,人表达SA2-6Gal受体为主,故人不易感染禽流感病毒和禽类不易感染人流感病毒;猪在流感病毒生态系统中是重要"基因混合器",有助于新病毒株在人的流行,此外,流感病毒生态系统中间宿主的范畴可能还包括多种陆地禽类如鸡和鹌鹑.然而,最近研究结果表明人呼吸道除表达SA2-6Gal受体,下呼吸道也表达SA2-3Gal受体,为禽流感病毒从禽类跨种属传递给人提供分子学基础.本文就流感病毒唾液酸受体在生态系统的分布和作用的研究进展作一综述.  相似文献   

14.
抗H5N1禽流感病毒VHH抗体库的构建   总被引:1,自引:1,他引:0  
目的:构建抗H5N1禽流感病毒的小羊驼免疫噬菌体重链可变区抗体库(VHH型抗体库),为抗H5N1的VHH抗体筛选奠定基础。方法:利用H5N1灭活疫苗免疫小羊驼,一定免疫时间后测定小羊驼外周血清中抗体中和活性,分离其外周淋巴细胞,利用RT-PCR方法得到VHH抗体片段。通过优化连接和电转化方法,将足量VHH片段与pCANTAB5E连接后电转入大肠杆菌TG1,获得VHH抗体基因库;检测基因库库容以及多样性,并采用血凝抑制试验对噬菌体抗体库进行初步功能性鉴定。结果:利用H5N1灭活疫苗免疫小羊驼四次后,其外周血清中抗体血清抑制效价可达1∶2 560,构建的VHH抗体基因库库容可达3×108,随机挑选14个抗体基因克隆进行测序鉴定,结果显示均为独立克隆,表明所建抗体库多样性好。上述基因库经辅助噬菌体拯救后,得到抗H5N1的噬菌体VHH型抗体初级库,对初级库进行血凝抑制试验,结果呈阳性,表明初级库中存在具有潜在中和活性的抗H5N1抗体。结论:结果表明,已成功构建抗H5N1禽流感病毒的小羊驼免疫噬菌体重链抗体库,为进一步筛选抗H5N1禽流感的重链抗体打下良好基础,并为H5N1的早期临床诊断和治疗提供新的手段。  相似文献   

15.
高致病性禽流感H5N1病毒研究进展   总被引:1,自引:0,他引:1  
1997年8月发现首例人禽流感病例以来,不断有人禽流感病例发生,截至2007年3月1日,WHO报道的经实验室确证的H5N1禽流感感染者共277例,死亡167例,死亡率超过50%。目前WHO对禽流感的预警是3级,即是一种新的流感亚型,已经在人类中引起严重疾病,但还没有在人类中持续传播流行。本文就目前H5N1禽流感病毒来源,跨越物种传播的机制,致病力决定因素及人间传播能力等几个关键的基础问题研究进展进行综述。  相似文献   

16.
深圳地区甲1(H1N1)亚型流感病毒基因特性的研究   总被引:4,自引:0,他引:4  
目的 了解近几年H1N1亚型毒株在深圳地区人群中活动加强及“O”相特性出现的分子生物学基础及其基因演变的特性。方法 病毒粒RNA经逆转录合成cDNA,用PCR扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定并推导出其所编码的氨基酸序列。进化树分析用DNASTAR公司出品的序列分析软件,MegAlign(103版)的Editseq(369版)。结果 根据病毒粒HA1基因特性,至少1995年以来深圳地区人群中同时流行着基因特性不同的三系毒株;1995~1997年H1N1毒株与A新加坡686(H1N1)病毒相比较,其HA1蛋白分子上第54和155位上分别插入和缺失一个糖基化点,同时氨基酸序列发生了替换。结论 近年来深圳地区人群中同时流行着HA1基因不同的三系H1N1亚型毒株。由于HA1蛋白分子上氨基酸序列发生了替换,尤其糖基化位点插入和缺失,造成1995年以来H1N1毒株活动加强,这些可能与毒株“O”相特性再现密切相关。  相似文献   

17.
目的探索Ⅰ型Na /H 交换器(NHE1)在脂多糖(LPS)引起的大鼠肺微血管内皮细胞(PMVECs)损伤中的作用。方法培养大鼠PMVECs。MTT比色试验检测细胞活力;比色法测试上清液中乳酸脱氢酶(LDH)活力和丙二醛(MDA)浓度;BECEF-AM荧光探针染色细胞,激光共聚焦显微镜下观察细胞内酸碱度,检测NHE1的活性;PT-PCR技术检测NHE1的mRNA表达。结果LPS不仅使细胞活力降低35%(P<0.05),使上清液中LDH和MDA分别升高3倍和2.5倍(P<0.05),而且使NHE1的活性增强,mRNA表达增多(P<0.05)。但是NHE1的抑制剂———环己阿米洛利(HMA)能显著抑制LPS引起的这些变化(P<0.05)。结论NHE1的活性增强和表达增多是LPS引起大鼠PMVECs损伤的重要机制。  相似文献   

18.
目的 探讨北京市中、小学生甲型H1N1流感感染的相关影响因素.方法采用1∶2病例-对照研究方法,通过问卷调查,在6个区县收集304对病例和对照的个人卫生习惯、疫苗接种史、搭乘交通工具等信息.结果多因素logistic回归结果显示,教室内学生密度,教室通风情况,课间活动范围,打喷嚏后是否洗手,睡眠时间,上学交通方式,接种...  相似文献   

19.
目的 探讨北京市中、小学生甲型H1N1流感感染的相关影响因素.方法采用1∶2病例-对照研究方法,通过问卷调查,在6个区县收集304对病例和对照的个人卫生习惯、疫苗接种史、搭乘交通工具等信息.结果多因素logistic回归结果显示,教室内学生密度,教室通风情况,课间活动范围,打喷嚏后是否洗手,睡眠时间,上学交通方式,接种甲流疫苗和不带病上课与甲型H1N1流感感染有关.结论参加户外活动、加强教室通风、注意手卫生、保证睡眠允足、不乘坐密闭的交通工具、接种甲型H1N1流感疫苗是预防中小学生甲型H1N1流感感染的重要手段.  相似文献   

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