H5N1重组禽流感病毒样颗粒免疫原性研究

Immunogenicity study of H5N1 avain influenza virus-like particles

目的对构建的H5N1重组禽流感病毒样颗粒(VLPs)进行初步免疫原性探讨,并与H5N1全病毒灭活疫苗(WIV)进行体液免疫和细胞免疫的比较。方法在0周和3周分别以纯化H5N1重组禽流感病毒样颗粒、H5N1全病毒灭活疫苗及pH7.2 PBS腿部肌肉注射BALB/c小鼠,于不同时间收集血清,以血凝抑制试验(HI)和血清IgG抗体酶联免疫吸附试验(ELISA)评估体液免疫,CD4+、CD8+T细胞亚群及酶联免疫斑点试验(ELISPOT)评估细胞免疫,并以同型毒株滴鼻攻击,观察小鼠存活率。结果病毒样颗粒各组和全病毒灭活疫苗免疫后小鼠血清ELISA IgG效价均有升高;中和抗体效价除病毒样颗粒120 ng/只免疫剂量外其他免疫小鼠HI效价均达1︰40;小鼠脾CD4+T淋巴细胞亚群分类:全病毒灭活疫苗组(600μg/只)为36.56%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为26.58%,32.20%,29.25%;PBS组为26.65%;CD8+T淋巴细胞亚群分类:全病毒灭活疫苗组(600 ng/只)为10.78%;病毒样颗粒组(120 ng/只,600 ng/只,2 500 ng/只)分别为1 3.53%,14.24%,1 3.35%;PBS组为10.69%。ELISPOT试验统计学数据显示,病毒样颗粒和全病毒灭活疫苗的小鼠脾单个核细胞分泌IFN-γ细胞与PBS组有显著性差异;小鼠保护性试验结果显示,除病毒样颗粒120 ng/只免疫剂量小鼠的存活率为87.5%外,其他病毒样颗粒实验组小鼠均为100%,PBS对照组为12.5%。结论 H5N1重组禽流感病毒样颗粒能诱导体液免疫和细胞免疫,并能抵御同型病毒株的攻击,可作为H5N1人用禽流感的候选疫苗。

Objective To study the immunogenic charactoristics of H5N1 avain influenza virus-like particle and compare the humoral immunity and cellular immunity elicited by H5N1 avain influenza virus-like particle and whole inactive H5N1 avain influenza virus. Methods BALB/c mice were immunized intramuscularly in legs with different HA doses of virus-like particles and whole inactive influenza virus, and then a boost dose given at 3 weeks after the primary dose, the humoral immunity responses were assessed by ELISA and hemagglutination inhibition （ H1 ） test, the cellular inununity was assessed by the relative amount of CD4^＋ or CD8^＋ cells and ELISPOT test. The immunized mice were challenged with lethal strain. Results The IgG antibody titer induced by virus-like particles and whole inactive virus can be all achieved, neutralizing antibody titer induced by virus-like particles can be reached to 1 ： 40 expect the 120 ng dose group. The Classification results of spleen T lymphocyte are as follows： （1） CD4^＋ T lymphocytes account for 36.56% in the whole inactive virus vaccine group, CD4^＋ T cells account for 26.58% , 32.20% and 29.25% respectively, in the virus-like particles （ 120 ng, 600 ng, 2 500 ng respectively ） groups, CD4^＋T cells accunt for 26.65% in PBS control group; （2） CD8^＋ T cells accunt for 10.78% in the whole inactive virus group, CD8^＋ T cells accunt for 13.53%, 14.24% and 13.35% respectively, in the virus-like particles （120 ng, 600 ng, 2 500 ng respectively）groups. CD8^＋T cells account for 10.69% in PBS control group. ELISPOT test resuhs showed that the number of IFN-γ secreting mononuclear cell in the virus-like particles group and whole inactive virus group is higher, there are significant differences between immune groups and PBS control group. The mice protection test results indicated that the protective rate is 87.5% only in the 120 ng HA dose of virus-like particle group. In the other groups the protective rates are 100%. In PBS control group it is 12.5%. Conclusions H5N1 avian influenza virus-like particles can induce humoralimmunity and cellular immunity and provide the protective effects against the challenge with wild virus strains, it can be used as an avain influenza vaccine for human use.