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
Gayathri Bommakantia, Michael P. Citronb, Robert W. Heplerb, Cheryl Callahanb, Gwendolyn J. Heideckerb, Tariq Ahmad Najara, Xianghan Lub, Joseph G. Joyceb, John W. Shiverb, Danilo R. Casimirob, Jan ter Meulenb, Xiaoping Liangb,1, and Raghavan Varadarajana,c,1
+ Author Affiliations

aMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India;
bMerck Research Laboratories, West Point, PA 19486;
cChemical Biology Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur P.O., Bangalore 560 064, India
Communicated by Peter S. Kim, Merck Research Laboratories, North Wales, PA, June 10, 2010 (received for review December 11, 2008)

Abstract
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.

hemagglutinin protein design bacterial expression

Footnotes
1To whom correspondence may be addressed. E-mail: varadar@mbu.iisc.ernet.in or xiaoping_liang@merck.com. Author contributions: G.B., R.W.H., J.G.J., J.W.S., D.R.C., J.t.M., X. Liang, and R.V. designed research; G.B., M.P.C., R.W.H., C.C., G.J.H., T.A.N., and X. Lu performed research; G.B., M.P.C., R.W.H., C.C., G.J.H., T.A.N., X. Lu, J.G.J., J.W.S., D.R.C., J.t.M., X. Liang, and R.V. analyzed data; and G.B., J.G.J., D.R.C., J.t.M., X. Liang, and R.V. wrote the paper.
The authors declare no conflict of interest.

See Commentary on page 13563.

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