氢氧化铝佐剂与甲型肝炎病毒抗原吸附条件的优化及吸附产物免疫原性评价

Optimization for condition of adsorbing hepatitis A virus antigen with aluminum hydroxide adjuvant and evaluation of immunogenicity of adsorption products

目的优化氢氧化铝Al(OH)_3佐剂与甲型肝炎病毒(hepatitis A virus,HAV)抗原的吸附条件,并评价吸附产物的免疫原性。方法选取缓冲盐终摩尔浓度(0.01、0.02和0.04 mol/L)、pH(5.5、6.0、6.5、7.0、7.5、8.0)、氯化钠(NaCl)终摩尔浓度(0.075、0.13、0.15、0.17、0.45、0.75、1.0 mol/L)、混合方式(稀释后混合、不稀释直接混合)及滴加顺序[HAV原液滴加至Al(OH)_3溶液、Al(OH)_3溶液滴加至HAV原液]进行单因素筛选。以Al(OH)_3吸附率为评价指标,通过正交试验对筛选出的因素进行优化。采用最优及次优组合条件制备的吸附产物经腹腔免疫NIH小鼠(雌雄各半),0.5 mL/只,每组10只,于免疫后28 d,经眼球或心脏采血,分离血清,HAV抗体定性检测试剂盒(ELISA法)检测HAV抗体水平,小鼠血清抗体阳转率评价吸附产物的免疫原性。结果单因素筛选结果表明,缓冲盐终摩尔浓度、pH、NaCl终摩尔浓度对吸附效果可产生明显影响(F分别为6.582、14.007、8.612,P均<0.05),混合方式和滴加顺序对吸附效果无影响(t分别为-0.696和1.153,P均>0.05),确定缓冲盐终摩尔浓度为0.01及0.02 mol/L,pH为5.5~7.0、NaCl终摩尔浓度为0.13~0.17 mol/L。正交试验确定影响吸附效果因素依次为:缓冲盐终摩尔浓度>pH>NaCl终摩尔浓度,最优组合为:pH 6.3+缓冲盐终摩尔浓度0.01 mol/L+NaCl终摩尔浓度0.15 mol/L,次优组合为:pH 5.8+缓冲盐终摩尔浓度0.02 mol/L+NaCl终摩尔浓度0.17 mol/L。最优及次优组合制备吸附产物免疫小鼠的血清抗体阳转率均为100%。结论优化条件制备的吸附产物具有良好的免疫原性,本研究为甲肝灭活疫苗研究奠定了基础。

Objective To optimize the adsorption condition of hepatitis A virus(HAV)antigen by aluminum hydroxide[Al(OH)_(3)]adjuvant and evaluate the immunogenicity of the adsorbed products.Methods The final molar concentration of buffer(0.01,0.02 and 0.04 mol/L),pH value(5.5,6.0,6.5,7.0,7.5 and 8.0),final molar concentration of sodium chloride(NaCl)(0.075,0.13,0.15,0.17,0.45,0.75 and 1.0 mol/L),mixing mode(mixing after dilution and mixing directly without dilution)and dropping order[HAV stock solution into Al(OH)_(3) solution,and Al(OH)_(3) solution into HAV stock solution]were selected for single factor screening.Taking Al(OH)_(3) adsorption rate as the evaluation index,the selected factors were optimized by orthogonal test.NIH mice were immunized intraperitoneally with adsorbed products prepared under optimal and suboptimal combination conditions with 0.5 mL/mouse,10 mice in each group with half male and half female.After 28 d of immunization,the blood samples were collected from eyeball or heart,and the serum was separated.The HAV antibody level was detected by ELISA,and the immunogenicity of adsorbed products was evaluated by the seroconversion rate of mouse serum antibody.Results The results of single factor screening test showed that the final molar concentration of buffer,pH value and final molar concentration of NaCl had significant influence on the adsorption effect(F=6.582,14.007 and 8.612,respectively,each P<0.05),while the mixing mode and dropping order had no effect(t=-0.696 and 1.153,respectively,each P>0.05).The final molar concentration of buffer was 0.01 and 0.02 mol/L,the pH value was 5.5~7.0,and the final molar concentration of NaCl was 0.13~0.17 mol/L.The orthogonal test showed that the order of the influence on adsorption effect was the final molar concentration of buffer>pH value>NaCl final molar concentration.The optimal combination was pH 6.3+final molar concentration of buffer 0.01 mol/L+NaCl molar final concentration 0.15 mol/L,and the suboptimal combination was pH 5.8+final molar concentration of buffer 0.02 mol/L+NaCl final molar concentration 0.17 mol/L.The seroconversion rate of serum antibody in mice immunized with the adsorbed products prepared by the optimal and suboptimal combination was both 100%.Conclusion The adsorbed products prepared under the optimized conditions have good immunogenicity,which lays a foundation of the research of inactivated hepatitis A vaccine.