Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge
Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge
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DOI：
10.1073/pnas.1007465107
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发表时间：
2010-08-03
影响因子：
11.1
通讯作者：
Varadarajan, Raghavan
中科院分区：
文献类型：
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作者：
Bommakanti, Gayathri;Citron, Michael P.;Varadarajan, Raghavan
流感病毒HA是感染过程中中和抗体的主要靶点，其序列在免疫压力下会发生遗传漂变和转移。HA的受体结合HA 1亚基相对于亚稳定的融合活性HA 2亚基显示出高得多的序列变异性，这可能是因为中和抗体在自然感染中主要针对前者。我们设计了一种基于HA 2的免疫原，使用蛋白质最小化方法，该方法结合了设计的突变以使HA 2的低pH构象不稳定。在大肠杆菌中表达所得到的构建体（HA 6），并从包涵体重折叠。蛋白质的生物物理学研究和突变分析表明，它被折叠成所需的中性pH构象，能够结合广泛中和的HA 2定向单克隆12 D1，而不是在以前的研究中观察到的低pH构象。HA 6在小鼠中具有高度免疫原性，并且小鼠被同源A/HK/68小鼠适应病毒保护免于致死性攻击。来自另一种H3株（A/Phil/2/82）的HA 6样构建体也保护小鼠免受A/HK/68攻击。包括在HA 6中的区域在亚型内高度保守，并且在进化枝内相当好地保守。用细菌产生的免疫原靶向高度保守的HA 2亚基是一种疫苗策略，可能有助于预防大流行。
Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.