tetano
Editor, Senior Moderator
Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines
Yuan Lua,
John P. Welsha, and
James R. Swartza,b,1
Author Affiliations
Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved November 15, 2013 (received for review May 7, 2013)
Significance
The discovery of neutralizing antibodies that block influenza infection by binding to the hemagglutinin (HA) stem domain raised the hope for broadly protective vaccines. These could avoid the need for annual vaccinations and reduce pandemic threats, and the stem subdomain of the trimeric HA ectodomain would be an ideal antigen. However, its production has proven extremely difficult. Here, we describe a simple procedure resulting in high yields of HA stem trimer recognized identically by a panel of neutralizing antibodies as compared with recognition of the full-length HA ectodomain. Cell-free protein synthesis is followed by a simple refolding procedure to produce a rationally mutated stem in which newly exposed protein surfaces are modified and trimerization is induced and covalently stabilized.
Abstract
The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.
http://www.pnas.org/content/111/1/125.abstract
Yuan Lua,
John P. Welsha, and
James R. Swartza,b,1
Author Affiliations
Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved November 15, 2013 (received for review May 7, 2013)
Significance
The discovery of neutralizing antibodies that block influenza infection by binding to the hemagglutinin (HA) stem domain raised the hope for broadly protective vaccines. These could avoid the need for annual vaccinations and reduce pandemic threats, and the stem subdomain of the trimeric HA ectodomain would be an ideal antigen. However, its production has proven extremely difficult. Here, we describe a simple procedure resulting in high yields of HA stem trimer recognized identically by a panel of neutralizing antibodies as compared with recognition of the full-length HA ectodomain. Cell-free protein synthesis is followed by a simple refolding procedure to produce a rationally mutated stem in which newly exposed protein surfaces are modified and trimerization is induced and covalently stabilized.
Abstract
The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.
http://www.pnas.org/content/111/1/125.abstract