H5N1型流感病毒神经氨酸酶在小鼠模型中的免疫保护性评价

Evaluation of immune protection of H5N1 influenza virus neuraminidase in mouse model

目的探讨在小鼠模型中H5N1型流感病毒神经氨酸酶(neuraminidase,NA)的免疫原性,并进行攻毒试验评价其免疫保护作用。方法制备H5N1型流感病毒裂解疫苗原液,磁珠法纯化获取NA蛋白。将BALB/c小鼠分为2μg NA、10μg NA、50μg NA、2μg NA+MF59、10μg NA+MF59、50μg NA+MF59和MF59组,共7组,每组10只,肌内注射免疫。初次免疫2周后,相同程序/剂量进行加强免疫,第4周采血分离血清,ELISA法测定效价;第5周进行攻毒试验,监测攻毒后2周内小鼠体重和体温变化,同时观察临床症状。结果磁珠法纯化的H5N1 NA纯度达90%,浓度为201.23μg/mL。2次免疫后,各剂量组血清效价呈剂量依赖性,加MF59佐剂组血清抗体效价均高于未加MF59佐剂组,其中50μg NA+MF59组效价最高(平均效价为121600)。攻毒试验发现,50μg NA+MF59能够保护100%(8只)的小鼠免受H5N1攻击,攻毒后50μg NA+MF59组临床评分低于MF59组,但两组小鼠体温变化差异无统计学意义(P>0.05)。结论免疫H5N1 NA能够诱导BLAB/c小鼠产生NA特异性抗体,使用MF59佐剂能够显著提高抗体效价,保护小鼠免受半数致死剂量的H5N1攻击,并能明显减轻临床症状。

Objective To investigate the immunogenicity of neuraminidase(NA)of H5N1 influenza virus in mouse model and evaluate the immune protection by challenge test.Methods The bulk of H5N1 influenza virus was prepared,from which NA protein was purified with magnetic beads.BALB/c mice were divided into seven groups,ten for each,and injected i.m.with 2μg NA,10μg NA,50μg NA,2μg NA+MF59,10μg NA+MF59,50μg NA+MF59 and MF59 respectively,and boosted according to the same schedules at the same dosages 2 weeks later.Serum samples were collected at week 4 after the first dose and determined for titer by ELISA.The mice were challenged at week 5 after the first dose and observed for the changes of bodyweight and body temperature as well as clinical symptoms within 2 weeks.Results The purity of H5N1 NA purified with magnetic beads reached 90%,while the concentration was 201.23μg/mL.After immunization for 2 times,the serum titers of various groups were dose-dependent.However,the serum antibody titers of mice immunized with NA containing MF59 adjuvant were higher than those containing no MF59 adjuvant,of which that in 50μg NA+MF59 group was the highest(with a mean of 121600).Challenge test showed that 50μg NA+MF59 protected 100%of the eight mice against challenge with H5N1 virus.The clinical score of 50μg NA+MF59 group after challenge was significantly lower than that of MF59 group,while the change of body temperatures in the two groups showed no significant difference(P>0.05).Conclusion Immunization with H5N1 NA induced specific NA antibody in BALB/c mice,and MF59 adjuvant significantly increased the antibody titer,which protected BALB/c mice from challenge with H5N1 virus at median lethal dose and significantly relieved the clinical symptoms.