为增强H7N9流感病毒HA DNA疫苗的免疫效果, 本文构建了含有流感病毒NP的5个重复优势T表位或B表位(包括B1线性表位和B2构象表位)的表达质粒(简称NPT和NPB), 以小鼠为动物模型, 将NPT和NPB分别与H7N9流感病毒HA DNA混合免疫BALB/c小鼠1次, 21 d后用致死剂量(20 LD50)的H7N9流感病毒攻击小鼠。通过检测免疫后小鼠血清特异性抗体滴度和攻毒后的免疫保护性指标-存活率、肺部残余病毒滴度及体重丢失率, 评价表位质粒与HA DNA疫苗混合免疫对小鼠的保护效果。试验结果表明:用致死性H7N9流感病毒攻击小鼠后, HA DNA组小鼠全部死亡,HA+NPT免疫组小鼠存活率达到100%,HA+NPB1和HA+NPB2免疫组, 小鼠存活率分别为30%和75%;混合免疫HA+NPT后小鼠抗体滴度升高明显, 攻毒后小鼠体重丢失明显降低。综上表明, 含有5个NP优势T表位或B表位的表达质粒能增强H7N9流感病毒HA DNA疫苗的免疫效果, 混合免疫一次可以使小鼠得到较好的保护。HA DNA和表位疫苗的混合免疫, 节约了疫苗用量, 降低了免疫成本, 为流感病毒核酸疫苗的临床开发提供了一定的试验基础。

In order to enhance the immune effect of H7N9 influenza virus HA DNA vaccine, the expression plasmids containing five repetitive dominant T epitopes or B epitopes, including B1 linear epitope or B2 conformational epitope, of influenza virus NP were constructed (NPT and NPB for short). BALB/c mice were immunized with H7N9 influenza virus HA DNA in combination with NPT or NPB, respectively. Twenty-one days later, the mice were challenged with a lethal dose (20 LD50) of H7N9 influenza virus. The protective effects of NP epitope plasmid in combination with HA DNA vaccine were evaluated by detecting vaccine specific antibody titer in serum, survival rate, lung viral titer and body weight loss of mice. The results showed that all mice vaccinated with only HA DNA died after the mice were challenged with the lethal H7N9 influenza virus; the mice survival rate of HA+NPT group reached 100%; the survival rate of mice vaccinated with HA+NPB1 or HA+NPB2 groups was 30% and 75%, respectively. The levels of antibodies were increased in the mixed immunization group compared with the HA alone group. The antibody level of the mice was significantly elevated after HA+NPT mixed immunization. Their loss rates of body weight were significantly reduced, and the body weight recovered after 21 days of challenge. This study showed that the expression plasmid containing five NP-preferred T epitopes or B epitopes could enhance the immune effect of H7N9 influenza virus HA DNA vaccine, and mixed immunization of NP epitope plasmid and HA DNA vaccine could provide better protection against H7N9 influenza virus in mice experiments. The combination of HA DNA and epitope vaccine can save the vaccine dosage and reduce the cost of immunization, and provide a certain experimental basis for the clinical development of influenza DNA vaccine.