(205b) Production of the Trimeric Influenza Hemagglutinin Stem Domain for Potentially Broadly Protective Influenza Vaccines

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. Even though producing isolated domains of non-modular, 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) using Escherichia coli-based cell-free protein synthesis (CFPS) and a simple refolding protocol. The CFPS platform provides a rapid, scalable and cost-effective means to combat pandemic and epidemic threats when a rapid response with large amounts of proteins are required. Several residues in the HA stem domain were mutated or removed to facilitate folding and trimeric assembly. The T4 bacteriophage fibritin foldon placed at the C-terminus of the HA stem domain induces trimer formation. The protein refolding conditions were also optimized. pH and the use of the detergent emerged as the most important factors. Multiple preparations indicate high production yields and process consistency. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed using a commercial antibody, C179, that blocks influenza infection by binding to the HA stem domain trimer. This neutralizing antibody recognizes the new stem domain trimer with approximately 30 fold higher affinity relative to the full-length HA protein, most likely because of improved steric availability. Based on the insights gained, we believe that similar procedures can be rapidly developed for the other influenza HA stem domains needed for universal vaccine candidates. We also feel that this overall protocol provides a new paradigm for the rational design and evaluation of stable properly folded domains of other multimeric proteins.