人禽流感病毒H5N1多价重组痘苗病毒(天坛株)疫苗在小鼠中的免疫原性研究

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.     

含H5N1-HA基因重组腺病毒疫苗的构建及其诱导免疫应答的初步探讨

目的 构建包含H5N1-HA基因的重组腺病毒疫苗并探讨其免疫效果.方法 用Admax系统构建包含H5N1-HA基因的重组腺病毒疫苗,并用PCR、Western-Blot等方法对重组病毒疫苗进行鉴定;疫苗免疫小鼠后,通过HI实验和ELISPOT实验检测其体液免疫和细胞免疫反应,评价其免疫效果.结果 成功得到了含有H5N1-HA基因的重组腺病毒疫苗;基因表达鉴定表明,HA基因能够在细胞中进行表达;血凝抑制实验结果显示小鼠产生的针对HA抗体滴度在1:320和1:640之间;ELISPOT结果显示实验组和对照组(PBS)相比斑点数量差异有统计学意义(P<0.05),以上免疫结果表明重组腺病毒载体疫苗可以诱导小鼠产生良好的特异性体液和细胞免疫反应.结论 含H5NA-HA的重组腺病毒疫苗可以诱导小鼠产生良好的免疫反应,为研制人禽流感疫苗打下基础.     

含HIV-1 gag基因的重组腺病毒5型与35型嵌合病毒免疫效果研究

目的探讨含HIV-1 gag基因的重组腺病毒5型与35型嵌合病毒(rAd5/F35)在BALB/c小鼠中的免疫效果。方法PCR,间接免疫荧光鉴定HIV-1gag基因在重组腺病毒(rAd5/F35-mod.gag)的正确插入和体外细胞水平的表达,用rAd5/F35-mod.gag以不同的方式免疫BALB/c小鼠,ELISA检测小鼠血清中的p24特异性抗体,细胞内细胞因子染色法检测小鼠的特异性细胞毒性T淋巴细胞(CTL)应答。结果重组腺病毒rAd5/F35-mod.gag在体外细胞水平可以很好的表达目的基因gag;在小鼠体内重组腺病毒rAd5/F35-mod.gag可诱导特异性的CTL应答和血清IgG抗体反应,用rAd5/F35单独免疫2次,所诱发的CTL和血清IgG反应最强。但Ad5初次感染后,产生的抗Ad5抗体可以抑制rAd5/F35疫苗的特异性免疫反应。结论重组腺病毒rAd5/F35-mod.gag单独免疫可在小鼠体内诱导特异性的CTL应答和血清IgG抗体反应。只改变Ad5纤突蛋白Fiber,不能避免抗Ad5抗体的抑制作用。     

突变型乙肝病毒前C-C基因疫苗免疫BALB/C小鼠的研究

目的 了解乙肝病毒前C-C基因疫苗(VEC)的突变型VE2、VE4免疫BALB/c小鼠后诱发特异性体液和细胞免疫的效果.方法 分别用突变型和非突变型DNA疫苗免疫BALB/c小鼠,ELISA法检测小鼠抗HBc、抗HBelgG,免疫第28天取小鼠脾细胞用酶免疫斑点(ELISPOt)方法和CTL杀伤试验检测特异性细胞免疫功能.结果 VE2、VFA组抗HBe水平高于VEC组,抗HBc水平差异无统计学意义.3种质粒均能引发特异性细胞免疫反应,VE4、VE2强于VEC;联用VM7免疫后能使VEC、VE2、VE4特异性细胞免疫反应增强.结论 突变型前C-C基因疫苗在诱导BALB/c小鼠特异性体液和细胞免疫方面优于突变前.γ干扰素基因可作为基因佐剂增强HBV DNA疫苗诱导的特异性细胞免疫反应.     

表达HIV-1 B亚型env基因的质粒DNA及腺病毒载体疫苗的免疫原性研究

目的 构建表达中国B亚型HIV-1流行株env基因的DNA及重组腺病毒载体疫苗,将其用于预防或治疗HIV感染.方法 构建质粒DNA疫苗pVR-gp160及重组腺病毒载体疫苗rAdV-gp160.将这两种疫苗以不同的方式免疫BALB/c小鼠,分别采用ELISPOT方法 和ELISA方法 检测免疫小鼠中HIV-1 Gp120特异性细胞免疫反应及抗体反应.结果 DNA疫苗单独免疫及DNA疫苗初免/腺病毒疫苗加强免疫的联合免疫方案皆可诱导较高水平的Gp120特异性细胞免疫反应;而在体液免疫方面,各实验组产生的Gp120特异性抗体水平都较低.结论 所构建的DNA疫苗及rAdV疫苗能有效表达Gp160蛋白,并可有效激活机体的细胞免疫反应.     

CVB3腺病毒载体疫苗Ad/MDC-VP1与DNA疫苗联合应用的免疫效果

目的 构建重组腺病毒Ad/MDC-VP1,观察Ad/MDC-VP1与NA疫苗联合应用的免疫效果.方法 构建、包装重组腺病毒Ad/MDC-VP1并检测目的 蛋白的表达.BALB/c小鼠随机分为Ad/MDC-VP1、pcDNA3/MDC-VP1、pcDNA3/MDC-VPl+Ad/MDC-VP1和PBS 4组,肌肉注射免疫小鼠.用ELISA法和微量中和试验法分别检测血清柯萨奇病毒B3(CVB3)VP1 IgG和中和抗体滴度;CCK-8法检测淋巴细胞增殖活性和特异性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小鼠的存活率.结果 成功构建并包装了重组腺病毒Ad/MDC-VP1,检测到目的 蛋白的表达.peDNA3/MDC-VP1+Ad/MDC-VP1组血清CVB3 VP1 IsG滴度、淋巴细胞增殖指数、CTL杀伤活性和对小鼠的保护率明显高于其他各组(P<0.05),血清病毒滴度低于其他各组(P<0.05).结论 Ad/MDC-VP1与DNA疫苗联合应用能显著提高小鼠细胞和体液免疫应答.     

CVB3腺病毒载体疫苗Ad/MDC-VP1与DNA疫苗联合应用的免疫效果

目的 构建重组腺病毒Ad/MDC-VP1,观察Ad/MDC-VP1与NA疫苗联合应用的免疫效果.方法 构建、包装重组腺病毒Ad/MDC-VP1并检测目的 蛋白的表达.BALB/c小鼠随机分为Ad/MDC-VP1、pcDNA3/MDC-VP1、pcDNA3/MDC-VPl+Ad/MDC-VP1和PBS 4组,肌肉注射免疫小鼠.用ELISA法和微量中和试验法分别检测血清柯萨奇病毒B3(CVB3)VP1 IgG和中和抗体滴度;CCK-8法检测淋巴细胞增殖活性和特异性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小鼠的存活率.结果 成功构建并包装了重组腺病毒Ad/MDC-VP1,检测到目的 蛋白的表达.peDNA3/MDC-VP1+Ad/MDC-VP1组血清CVB3 VP1 IsG滴度、淋巴细胞增殖指数、CTL杀伤活性和对小鼠的保护率明显高于其他各组(P<0.05),血清病毒滴度低于其他各组(P<0.05).结论 Ad/MDC-VP1与DNA疫苗联合应用能显著提高小鼠细胞和体液免疫应答.     

CVB3腺病毒载体疫苗Ad/MDC-VP1与DNA疫苗联合应用的免疫效果

目的 构建重组腺病毒Ad/MDC-VP1,观察Ad/MDC-VP1与NA疫苗联合应用的免疫效果.方法 构建、包装重组腺病毒Ad/MDC-VP1并检测目的 蛋白的表达.BALB/c小鼠随机分为Ad/MDC-VP1、pcDNA3/MDC-VP1、pcDNA3/MDC-VPl+Ad/MDC-VP1和PBS 4组,肌肉注射免疫小鼠.用ELISA法和微量中和试验法分别检测血清柯萨奇病毒B3(CVB3)VP1 IgG和中和抗体滴度;CCK-8法检测淋巴细胞增殖活性和特异性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小鼠的存活率.结果 成功构建并包装了重组腺病毒Ad/MDC-VP1,检测到目的 蛋白的表达.peDNA3/MDC-VP1+Ad/MDC-VP1组血清CVB3 VP1 IsG滴度、淋巴细胞增殖指数、CTL杀伤活性和对小鼠的保护率明显高于其他各组(P<0.05),血清病毒滴度低于其他各组(P<0.05).结论 Ad/MDC-VP1与DNA疫苗联合应用能显著提高小鼠细胞和体液免疫应答.     

CVB3腺病毒载体疫苗Ad/MDC-VP1与DNA疫苗联合应用的免疫效果

目的 构建重组腺病毒Ad/MDC-VP1,观察Ad/MDC-VP1与NA疫苗联合应用的免疫效果.方法 构建、包装重组腺病毒Ad/MDC-VP1并检测目的 蛋白的表达.BALB/c小鼠随机分为Ad/MDC-VP1、pcDNA3/MDC-VP1、pcDNA3/MDC-VPl+Ad/MDC-VP1和PBS 4组,肌肉注射免疫小鼠.用ELISA法和微量中和试验法分别检测血清柯萨奇病毒B3(CVB3)VP1 IgG和中和抗体滴度;CCK-8法检测淋巴细胞增殖活性和特异性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小鼠的存活率.结果 成功构建并包装了重组腺病毒Ad/MDC-VP1,检测到目的 蛋白的表达.peDNA3/MDC-VP1+Ad/MDC-VP1组血清CVB3 VP1 IsG滴度、淋巴细胞增殖指数、CTL杀伤活性和对小鼠的保护率明显高于其他各组(P<0.05),血清病毒滴度低于其他各组(P<0.05).结论 Ad/MDC-VP1与DNA疫苗联合应用能显著提高小鼠细胞和体液免疫应答.     

CVB3腺病毒载体疫苗Ad/MDC-VP1与DNA疫苗联合应用的免疫效果

目的 构建重组腺病毒Ad/MDC-VP1,观察Ad/MDC-VP1与NA疫苗联合应用的免疫效果.方法 构建、包装重组腺病毒Ad/MDC-VP1并检测目的 蛋白的表达.BALB/c小鼠随机分为Ad/MDC-VP1、pcDNA3/MDC-VP1、pcDNA3/MDC-VPl+Ad/MDC-VP1和PBS 4组,肌肉注射免疫小鼠.用ELISA法和微量中和试验法分别检测血清柯萨奇病毒B3(CVB3)VP1 IgG和中和抗体滴度;CCK-8法检测淋巴细胞增殖活性和特异性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小鼠的存活率.结果 成功构建并包装了重组腺病毒Ad/MDC-VP1,检测到目的 蛋白的表达.peDNA3/MDC-VP1+Ad/MDC-VP1组血清CVB3 VP1 IsG滴度、淋巴细胞增殖指数、CTL杀伤活性和对小鼠的保护率明显高于其他各组(P<0.05),血清病毒滴度低于其他各组(P<0.05).结论 Ad/MDC-VP1与DNA疫苗联合应用能显著提高小鼠细胞和体液免疫应答.     

Protective Efficacy of the Conserved NP,PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD₅₀) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD₅₀ and 10 LD₅₀ and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics.     

Immunization with a low dose of hemagglutinin-encoding plasmid protects against 2009 H1N1 pandemic influenza virus in mice

A vaccine against the novel pandemic influenza virus (2009 H1N1) is available, but several problems in preparation of vaccines against the new emerging influenza viruses need to be overcome. DNA vaccines represent a novel and powerful alternative to conventional vaccine approaches. To evaluate the ability of a DNA vaccine encoding the hemagglutinin (HA) of 2009 H1N1 to generate humoral responses and protective immunity, BALB/c mice were immunized with various doses of 2009 H1N1 HA-encoding plasmid and anti-HA total IgG, hemagglutination inhibition antibodies and neutralizing antibodies were assayed. The total IgG titers against HA correlated positively with the doses of DNA vaccine, but immunization with either a low dose (10 μg) or a higher dose (25-200 μg) of HA plasmid resulted in similar titers of hemagglutination inhibition and neutralizing antibodies, following a single booster. Further, 10 μg plasmid conferred effective protection against lethal virus challenge. These results suggested that the DNA vaccine encoding the HA of 2009 H1N1 virus is highly effective for inducing neutralizing antibodies and protective immunity. DNA vaccines are a promising new strategy for the rapid development of efficient vaccines to control new emerging pandemic influenza viruses.     

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.     

Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD₅₀]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.     

Polygenic virulence factors involved in pathogenesis of 1997 Hong Kong H5N1 influenza viruses in mice

Virulence factors of influenza A (H5N1) viruses collected in 1997 from mammalian hosts were examined using a BALB/c mouse model. Fifteen amino acid (aa) residues in four influenza virus genes which correlated with high- and low-pathogenic phenotypes in mice were identified by analyzing sequence alignments. In addition to these specific residues, the effects of aa residue 627 of the PB2 gene, and the hemagglutinin (HA) and neuraminidase (NA) genes were also investigated using a reverse genetics system established with representative viruses of low (A/Hong Kong/486/97) and high (A/Hong Kong/483/97) pathogenicity for mice. None of 15 aa residues alone had any effect on virulence. The HA and NA genes had a synergistic effect on virulence and the absence of a glycosylation site at aa154 in the HA gene also increased virulence of virus. Multiple genes are involved in virulence of Hong Kong H5N1 influenza A viruses for mice with the presence of lysine at aa627 in the PB2 gene exhibiting a significantly larger effect than the HA and NA genes.     

Genetic analysis of influenza A/H3N2 and A/H1N1 viruses circulating in Vietnam from 2001 to 2006

Influenza A virus has the ability to overcome immunity from previous infections through the acquisition of genetic changes. Thus, understanding the evolution of the viruses in humans is important for the surveillance and the selection of vaccine strains. A total of 30 influenza A/H3N2 viruses and 35 influenza A/H1N1 viruses that were collected in Vietnam from 2001 to 2006 were used to analyze the evolution of the hemagglutinin (HA), neuraminidase (NA), and matrix protein (M) genes. Phylogenetic analysis of individual gene segments revealed that the HA and the NA genes of the influenza A viruses evolved in a sequential way. However, the evolutionary pattern of the M gene proved to be nonlinear and was not linked with that of the HA and NA genes. Genetic drift in HA1 segments, especially in the antigenic sites of A/H3N2 viruses, occurred more frequently in A/H3N2 viruses than it did in A/H1N1 viruses. Two reassortants, one influenza A/H3N2 strain and one A/H1N1 strain, were found on the basis of the phylogenetic analysis of the three genes. While both genetic mutation and reassortment contributed to their evolution, the frequency of genetic changes and reassortment events differs between the two subtypes. As influenza viruses circulate throughout the year, we emphasize the importance of surveillance in tropical and subtropical zones, where the emergence of new strains may be detected earlier than it is in temperate zones.     

Recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus partially protects mice from challenge with heterologous virus: A/HK/1/68 (H3N2)

Summary.  Immunization with recombinant adenoviral vaccine that induces potent immunity has been applied to many infectious diseases. We report here developing a recombinant adenoviral vaccine encoding the HA gene from swine H3N2 influenza virus (SIV). Two replication-defective recombinant adenoviruses were generated: (1) rAd-HA: recombinant adenovirus encoding the HA gene from swine H3N2 influenza virus, and (2) rAd-vector: a control recombinant adenovirus containing adenovirus and transfer plasmids without a foreign HA gene. Mice given rAd-HA developed high titers of neutralizing and hemagglutination inhibition antibodies to SIV in comparison to mice inoculated with rAd-vector or PBS as early as 2 weeks after immunization, and these antibodies were substantially increased in the mice given rAd-HA within the next 3 weeks following the first dose. However, these antibodies were not able to neutralize the virus, A/HK/68 (H3N2), used for challenge. Nonetheless mice immunized with one or two doses of rAd-HA were protected from lethal challenge with heterologous virus, A/HK/1/68 (H3N2). A statistically significant (P < 0.03) difference between survival rates of rAd-HA mice vs. rAd-vector or PBS mice was observed. Received April 2, 2002; accepted June 18, 2002