柯萨奇病毒B3的VP1 DNA疫苗与蛋白或重组腺病毒疫苗不同组合免疫方式的免疫效果观察

目的 观察柯萨奇病毒B3 (Coxsackievirus B3, CVB3)衣壳蛋白VP1 DNA疫苗初免后VP1蛋白或重组腺病毒rAd/VP1加强的prime-boost策略的免疫效果。方法 用CVB3 VP1的真核表达质粒pcDNA3/VP1初次免疫小鼠后，分别用VP1蛋白或重组腺病毒rAd/VP1加强免疫2次。检测免疫小鼠血清特异性IgG抗体、中和抗体滴度以及脾脏细胞毒性T淋巴细胞(cytotoxic lymphocytes，CTLs)杀伤活性；致死量CVB3攻击后，检测小鼠血中病毒滴度并观察动物的存活情况。结果 pcDNA3/VP1 +VP1蛋白组小鼠血清IgG抗体、中和抗体滴度以及动物生存率明显高于pcDNA3/VP1 + rAd/VP1组和pcDNA3/VP1质粒组（P<0.05）； pcDNA3/VP1 + rAd/VP1组和pcDNA3/VP1 +VP1蛋白组小鼠脾脏CTLs杀伤活性明显高于pcDNA3/VP1 质粒组（P<0.05）。结论 质粒pcDNA3/VP1初次免疫后，VP1蛋白或重组腺病毒rAd/VP1加强的prime-boost策略有较好的免疫效果，两者比较pcDNA3/VP1 +VP1蛋白prime- boost免疫策略的效果更为明显。

Generation of protective immune responses against coxsackievirus B3 challenge by DNA priming VP1 protein or recombinant adenovirus boosted vaccination

In order to compare the immune effects of two different prime-boost strategies, BALB/c mice were primed immunization with eukaryotic expressed plasmid pcDNA3/VP1 contained the VP1 capsid gene of Coxsackievirus B3 (CVB3), followed by two boosts with the VP1 protein from procaryotic cells or recombinant adenovirus rAd/VP1. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. Enzyme-linked immunosorbent assay 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 lymphocytes (CTLs) killing activity of spleen lymphocytes was detected with Cytotoxicity Counting Kit-8 assay. Followed the third immunization, mice were challenged with CVB3 infection. Virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose assay on HeLa cell monolayers. Percentage of animals surviving was observed after lethal CVB3 attack over a period of 21 days. The results showed the titers of specific IgG antibody and neutralizing antibody in sera of the pcDNA3/VP1 and VP1 protein regime immunized mice were the highest(P<0.05) among the three groups including pcDNA3/VP1, pcDNA3/VP1 and VP1 protein , pcDNA3/VP1 and rAd/VP1 groups. CTLs killing activity of spleen lymphocytes of the pcDNA3/VP1 and rAd/VP1 immunized mice were higher (P<0.05) than the pcDNA3/VP1 or pcDNA3/VP1 and VP1 protein group. In addition, the pcDNA3/VP1 and VP1 protein vaccine resulted in stronger protection mice from lethal CVB3 challenge and a lower significant reduction of viral load in sera of immunized mice after acute CVB3 infection. It had been concluded pcDNA3/VP1 priming and VP1 protein or rAd/VP1 boosted immunization had obvious immune responses against CVB3 infection in mice. The pcDNA3/VP1 priming and VP1 protein boosted regimen surpassed plasmid pcDNA3/VP1 and recombinant adenovirus rAd/VP1 regime and might be a promising vaccine candidate against CVB3 infection.