B型流感病毒核酸疫苗和CpG基序免疫增强作用研究
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摘要
本论文介绍了B型流感病毒DNA疫苗以及CpG基序作为佐剂对其免疫增强效应的研究。
血凝素(Hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)是B型流感病毒两种最重要的抗原蛋白,本文首先探讨了一次接种B型流感病毒(B/Ibaraki/2/85)DNA疫苗对BALB/c小鼠的免疫保护作用。分别采用不同剂量的HA DNA疫苗(100μg、50μg、10μg、5μg、1μg),NA DNA疫苗(50μg、10μg、5μg、1μg),以及HA和NA联合DNA疫苗(10μg、5μg、1μg),一次接种BALB/c小鼠。接种后四周,用致死量流感病毒(B/Ibaraki/2/85)攻击小鼠,病毒攻击后三天,取血清检测抗-HA和抗-NA抗体。同时测定小鼠肺部病毒含量,观察记录小鼠存活率和体重丢失情况。结果发现:100μg HA DNA疫苗,10μg NA DNA疫苗以及5μg联合DNA疫苗(HA+NA)接种的小鼠全部存活。结合小鼠肺部病毒含量和体重变化数据,得出以下结论:一次接种DNA疫苗可以为小鼠提供完全有效的保护,联合DNA疫苗免疫保护作用强于单一DNA疫苗,NA DNA疫苗免疫保护作用略强于HA DNA疫苗。
为了探讨CpG基序作为免疫佐剂的效应,本研究构建了包含CpG基序的质粒:pcD3d(+CpG)HA和不包含CpG基序的质粒pcD3d(-)HA。并采用不同的剂量,不同的免疫次数(一次或两次),分别免疫BALB/c小鼠,初免后四周(或加强免疫后一周),用致死量流感病毒(B/Ibaraki/2/85)攻击小鼠,病毒攻击后三天,取血清检测抗-HA IgG抗体,并观察小鼠存活率和体重变化情况。结果显示,含CpG基序的DNA疫苗能够进一步提高抗体水平,改善小鼠病毒攻击后的临床体症。实验表明CpG基序能有效提高小鼠抗B型流感病毒攻击的能力。
In this study, various doses of HA DNA and NA DNA were inoculated alone or as a mixture into mice once by electroporation. Four weeks after immunization, the mice were challenged with a lethal dose of influenza B virus. The induced serum IgG antibody levels and the survival rates of mice were measured to evaluate the immunity efficacy of the DNA vaccines. The results showed that a single dose of 100μg HA DNA or 10μg NA DNA provided 100% protection in mice against the lethal challenge. Moreover, when the two plasmid DNAs were coinoculated into mice, a single dose of 5μg of tne two plasmids each provided 100% protection. These indicate that a single immunization of HA DNA, NA DNA or a mixture of HA DNA and NA DNA would be an efficient method in preventing influenza B virus infection.To investigate the immune response induced by one or two times of inoculation with influenza B virus HA DNA vaccine and the adjuvant effect of CpG motif, BALB/c mice were immunized i.m. by electroporation once or twice (at a 3- week interval ) with plasmid HA DNA at different doses. Four weeks after the first inoculation (or one week after the second inoculation), the mice were challenged with a lethal dose of influenza virus (B/Ibaraki/2/85). The experiments showed that: 1) mice immunized with a single dose of 100μg HA DNA all
血凝素(Hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)是B型流感病毒两种最重要的抗原蛋白,本文首先探讨了一次接种B型流感病毒(B/Ibaraki/2/85)DNA疫苗对BALB/c小鼠的免疫保护作用。分别采用不同剂量的HA DNA疫苗(100μg、50μg、10μg、5μg、1μg),NA DNA疫苗(50μg、10μg、5μg、1μg),以及HA和NA联合DNA疫苗(10μg、5μg、1μg),一次接种BALB/c小鼠。接种后四周,用致死量流感病毒(B/Ibaraki/2/85)攻击小鼠,病毒攻击后三天,取血清检测抗-HA和抗-NA抗体。同时测定小鼠肺部病毒含量,观察记录小鼠存活率和体重丢失情况。结果发现:100μg HA DNA疫苗,10μg NA DNA疫苗以及5μg联合DNA疫苗(HA+NA)接种的小鼠全部存活。结合小鼠肺部病毒含量和体重变化数据,得出以下结论:一次接种DNA疫苗可以为小鼠提供完全有效的保护,联合DNA疫苗免疫保护作用强于单一DNA疫苗,NA DNA疫苗免疫保护作用略强于HA DNA疫苗。
为了探讨CpG基序作为免疫佐剂的效应,本研究构建了包含CpG基序的质粒:pcD3d(+CpG)HA和不包含CpG基序的质粒pcD3d(-)HA。并采用不同的剂量,不同的免疫次数(一次或两次),分别免疫BALB/c小鼠,初免后四周(或加强免疫后一周),用致死量流感病毒(B/Ibaraki/2/85)攻击小鼠,病毒攻击后三天,取血清检测抗-HA IgG抗体,并观察小鼠存活率和体重变化情况。结果显示,含CpG基序的DNA疫苗能够进一步提高抗体水平,改善小鼠病毒攻击后的临床体症。实验表明CpG基序能有效提高小鼠抗B型流感病毒攻击的能力。
In this study, various doses of HA DNA and NA DNA were inoculated alone or as a mixture into mice once by electroporation. Four weeks after immunization, the mice were challenged with a lethal dose of influenza B virus. The induced serum IgG antibody levels and the survival rates of mice were measured to evaluate the immunity efficacy of the DNA vaccines. The results showed that a single dose of 100μg HA DNA or 10μg NA DNA provided 100% protection in mice against the lethal challenge. Moreover, when the two plasmid DNAs were coinoculated into mice, a single dose of 5μg of tne two plasmids each provided 100% protection. These indicate that a single immunization of HA DNA, NA DNA or a mixture of HA DNA and NA DNA would be an efficient method in preventing influenza B virus infection.To investigate the immune response induced by one or two times of inoculation with influenza B virus HA DNA vaccine and the adjuvant effect of CpG motif, BALB/c mice were immunized i.m. by electroporation once or twice (at a 3- week interval ) with plasmid HA DNA at different doses. Four weeks after the first inoculation (or one week after the second inoculation), the mice were challenged with a lethal dose of influenza virus (B/Ibaraki/2/85). The experiments showed that: 1) mice immunized with a single dose of 100μg HA DNA all
引文
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[3] Yao Yongxiu, Louise J M, McCauley J W, et al. Sequences in Influenza A Virus PB_2 Protein that Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines [J]. J Virol, 2001, 75 (11) : 5410-15.
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[6] Kim HW, Brandt CD, Arrobio JO, et al. Chanock RM, Parrott RH. Influenza A and B virus infection in infants and young children during the years 1957～1976 [J]. Am J Epidemiol, 1979, 109: 464～79.
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[14] Hall CE, Cooney MK, Fox J P. The Seattle virus watch. Ⅳ. Comparative epiderniologic observations of infections with influenza A and B viruses, 1965～1969, in families with Young children [J]. Am J Epidemiol, 1973, 98: 365-80.
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[17] Ohishi K, Ninomiya A, Kida H, et al. Serological evidence of transmission of human influenza A and B viruses to Caspian seals (Phoca caspica) [J]. Microbiol Immunol, 2002, 46 (9) : 639-44.
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