Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge

被引:184
作者
Bommakanti, Gayathri [2 ]
Citron, Michael P. [1 ]
Hepler, Robert W. [1 ]
Callahan, Cheryl [1 ]
Heidecker, Gwendolyn J. [1 ]
Najar, Tariq Ahmad [2 ]
Lu, Xianghan [1 ]
Joyce, Joseph G. [1 ]
Shiver, John W. [1 ]
Casimiro, Danilo R. [1 ]
ter Meulen, Jan [1 ]
Liang, Xiaoping [1 ]
Varadarajan, Raghavan [2 ,3 ]
机构
[1] Merck Res Labs, West Point, PA 19486 USA
[2] Indian Inst Sci, Mol Biophys Unit, Bangalore 560012, Karnataka, India
[3] Jawaharlal Nehru Ctr Adv Sci Res, Chem Biol Unit, Bangalore 560064, Karnataka, India
关键词
hemagglutinin; protein design; bacterial expression; HUMAN MONOCLONAL-ANTIBODY; A VIRUS; RECEPTOR-BINDING; MEMBRANE-FUSION; DISULFIDE BONDS; HEMAGGLUTININ; NEUTRALIZATION; EPITOPE; IDENTIFICATION; INFECTIVITY;
D O I
10.1073/pnas.1007465107
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:13701 / 13706
页数:6
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