Variation in the divalent cation requirements of influenza A virus N2 neuraminidases
Variation in the divalent cation requirements of influenza A virus N2 neuraminidases
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DOI：
10.1093/jb/mvg151
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发表时间：
2003-09-01
影响因子：
2.7
通讯作者：
Brett, IC
中科院分区：
文献类型：
--
作者：
Johansson, BE;Brett, IC
从1957 ~ 1994年的几种流感病毒候选疫苗株中，用层析法纯化了流感病毒N2神经氨酸酶。测定了各菌株的酶动力学参数和免疫原性。在离子强度保持不变的情况下，Ca2+或Mg2+增加了酶活性的初始速率。早期N2-NA菌株的初速度、V-max/K-m和V-max均最高。添加Ca2+或Mg2+前后各流感病毒株酶活性差异显著：添加二价阳离子前V-max/K-m变化范围为0.54 ~ 0.88 M-1 s(-1)， V-max变化范围为2.45 ~ 4.3 s(-1)；并且在添加外源Ca2+或Mg2+后，每个菌株的这些动力学参数都增加了大约2倍。EDTA彻底透析降低了每个菌株的初始速度，菌株之间存在显着差异，其范围为原始活性的0.1%至8%。通过添加外源性Ca2+或Mg2+，活性部分恢复，从透析前水平的8%到60%不等，但未达到原始率。测试菌株（即A/Japan/57和A/Johannesburgt94）的酶活性降低伴随着na免疫原性的平行降低，根据NI滴度测量，抗体应答降低高达76%，ELISA滴度降低高达68%。在透析后样品中添加Ca2+或Mg2+可使免疫原性恢复到透析前NI滴度的80%和透析前ELISA滴度的78%。通过酶动力学参数和免疫原性研究，透析对早期菌株的影响最小。Zn2+对所有菌株的活性均有轻微的抑制作用。回顾这些菌株的核酸序列不能预测它们的酶活性、免疫原性或对透析的反应。如果在流感疫苗接种中需要对神经氨酸酶免疫，那么疫苗候选菌株的选择不仅必须包括抗原变化分析和序列分析，还必须包括酶学研究和确定在生产过程中保持免疫原性和活性的二价阳离子的要求。
Influenza virus N2 neuraminidases were chromatographically purified from several vaccine candidate strains from 1957 to 1994. Enzymatic kinetic parameters and immunogenicity were tested for each strain. For each NA tested, with ionic strength held constant, Ca2+ or Mg2+ increased the initial rate of enzymatic activity. Earlier N2-NA strains had the highest initial velocity, V-max/K-m and V-max. There were significant differences among the influenza virus strains in enzymatic activity before and after addition of Ca2+ or Mg2+: V-max/K-m varied from 0.54 M-1 s(-1) to 0.88 M-1 s(-1) and V-max varied from 2.45 s(-1) to 4.3 s(-1) before the addition of a divalent cation; and increased approximately 2-fold each of these kinetic parameters for each strain after the addition of exogenous Ca2+ or Mg2+. Exhaustive dialysis with EDTA reduced the initial velocity of each strain with significant differences found among strains, with a range of 0.1% to 8% of original activity. Activity was partially restored by the addition of exogenous Ca2+ or Mg2+, varying from 8% to 60% of pre-dialysis levels, but original rates were not achieved. This reduction in enzymatic activity for the tested strains (i.e., A/Japan/57 and A/Johannesburgt94) was accompanied by a parallel decrease in NA-immunogenicity, with antibody response decreasing by as much as 76% as measured by NI titer, and ELISA titer decreasing by as much as 68%. The addition of Ca2+ or Mg2+ to the post-dialysis sample restored immunogenicity to as much as 80% of pre-dialysis NI titers and as much as 78% of pre-dialysis ELISA titers. Dialysis had the least effect on early strains as measured by enzymatic kinetic parameters and immunogenicity studies. Zn2+ had a slight inhibitory effect on the activity of all tested strains. Review of the nucleic acid sequence of each of these strains could not predict their enzymatic activity, immunogenicity or response to dialysis. If immunity against neuraminidase is desirable in vaccination against influenza, selection of vaccine candidate strains must include not only analysis of antigenic changes and sequence analysis but also enzymatic studies and determination of the requirement of divalent cations to maintain immunogenicity and activity during production.