一种狂犬病毒疫苗佐剂的制备及其免疫效果研究

Preparation of a Rabies Virus Vaccine Adjuvant and its Immune Effect Evaluation

目的:以角鲨烯、山梨醇、吐温为组分,在琥珀酸缓冲液中混和甲型副伤寒沙门菌鞭毛蛋白制备复合佐剂Nano-fla,评价该佐剂对人二倍体狂犬疫苗的免疫效果和安全性。方法:以人二倍体细胞制备的狂犬灭活疫苗为抗原,BALB/c小鼠设置PBS对照组、全剂量疫苗组、半剂量疫苗组、低剂量佐剂组(含半剂量抗原+5μg鞭毛蛋白)、中等剂量佐剂组(含半剂量抗原+10μg鞭毛蛋白),免疫程序为在0、3、7 d肌肉注射,并在首针免疫后的第7、14 d尾静脉采血分离血清,通过快速免疫荧光灶抑制实验检测中和抗体,通过酶联免疫斑点实验检测细胞因子水平。结果:首针免疫后第7 d,中等剂量佐剂组的中和抗体达保护效力水平,显著高于全剂量疫苗组和低剂量佐剂组;第14 d时中等剂量佐剂组的IgG抗体浓度显著高于全剂量疫苗对照组;第14 d中等剂量佐剂组与全剂量疫苗组相比,分泌IFN-γ和IL-4的淋巴细胞数量显著增加。结论:复合佐剂Nano-fla应用到狂犬疫苗中,能有效刺激小鼠体液免疫和细胞免疫应答,更早地产生中和抗体,且能有效降低抗原用量,具有潜在应用价值。

Objective:To prepare a complex adjuvant(named as Nano-fla)of Salmonella paratyphi A flagellin,squalene,sorbitol and Tween,and to evaluate its immune effect and preliminary safety in humans diploid rabies vaccine.Methods:The rabies inactivated vaccine prepared from human diploid cells was used as antigen.BALB/c mice were divided into PBS control group,full-dose vaccine group,half-dose vaccine group,low-dose group(containing half-dose antigen and 5 μg flagellin)and medium-dose adjuvant group(containing half-dose antigen and 10 μg flagellin),intramuscular injected on days 0,3,and 7 and blood was collected from the tail vein on the 7th and 14th day after the first immunization.Neutralizing antibodies were detected by immunofluorescence inhibition assay and cytokine levels were detected by enzyme-linked immunospot assay.Results:On the 7th day after the first injection,the neutralizing antibody in the medium dose adjuvant group reached the protective efficacy level,which was significantly higher than the full dose vaccine group and the low dose adjuvant group;on the 14th day,the IgG antibody concentration in the medium dose adjuvant group was significantly higher than the full-dose vaccine control group;the medium-dose adjuvant group showed a significant increase in the number of lymphocytes secreting IFN-γ and IL-4 compared to the full-dose vaccine group.Conclusion:Admistration with compound adjuvant Nano-fla can effectively stimulate the humoral and cellular immune responses of mice,produce neutralizing antibodies earlier,and effectively reduce the dose of antigen,showing its potential application value.