We have identified the structural elements necessary for efficient refolding of a protective HA subunit after recombinant protein expression in E.coli
. Structural information is available for the H1 
, H3 
, H7 
, H5 and H9 subtypes 
of influenza A as well as for an influenza C HEF (hemagglutinin, esterase, and fusion glycoprotein) 
. Comparisons of these structures show that the different HAs, while antigenically distinct, are structurally similar and share a common sub-domain organization. Each HA monomer folds to form a membrane proximal stalk and a membrane distal globular head domain. The globular head stands independently from the central stalk and contains the majority of the neutralizing antibody epitopes 
. We designed three PR8 HA prototypic constructs to test the hypothesis that secondary structures such as the β-pleated sheets near these linker peptides are sufficient to support the head domain as a self-stabilizing unit that can be engineered and expressed apart from the rest of the molecule.
We find that of the three prototypic constructs evaluated, STF2.HA1-2 provides the level of expression and ease of conformationally correct refolding required to support a truly efficient, scalable manufacturing process. This molecule expressed well in our prokaryotic system and refolded easily using rapid dilution. STF2.HA1-1 also refolded albeit with somewhat lower efficiency than STF2.HA1-2, presumably as a consequence of the additional domain and two additional disulfide bonds. Western blots of STF2.HA1-1 run under reducing and non-reducing conditions and probed with PR8 convalescent sera reveal a band in the non-reduced sample that co-migrates with the main band in the reduced sample suggesting that a significant proportion of the protein remained misfolded. STF2.HA1-3 refolded the least efficiently, most likely due to the absence of secondary structures required for stable refolding. Reactivity with a panel of defined neutralizing monoclonal antibodies further supports this conclusion.
STF2.HA1-2 (PR8) was found to be highly immunogenic and efficacious against a lethal challenge in the mouse model. Mice receiving doses of STF2.HA1-2 (PR8) as low as 0.3 µg were protected against a lethal challenge of virus. These data demonstrate that a subunit of HA based on the globular head domain can be fully protective in a standard mouse lethal challenge model. When the same principles of design were applied to the currently circulating seasonal strain, A/Solomon Islands/3/2006, we found that STF2.HA1-2 (SI) was highly immunogenic in both mice and rabbits. In mice doses of 1 µg elicited geometric mean neutralizing titers of 1
297 and in rabbits doses of 5 µg elicited titers of 1
453. Thus, the principles of design for these protective subunit vaccines can be applied to different HA molecules.
A key benefit with this approach is that the STF2.HA1-2 recombinant fusion protein can be made quickly, inexpensively and in quantities sufficient to meet global needs. The efficiency of this technology translates approximately into a 1,000 fold gain in production. As a point of reference, the average yield for cell culture is 3 mg/L; for egg based production, 7 mg/L; for baculovirus recombinant synthetic protein, 13 mg/L and for the standard prokaryotic system described here, 3,700 mg/L. This increase in production capacity, along with the fact that it is carried out in a prokaryotic system, provides an opportunity to address several shortcomings of the current egg-based system. One advantage deriving from increased capacity is the ability to increase the dose of antigen. Studies have shown that persons greater than 65 years of age respond less well to the standard vaccine, and that increasing the dose of HA four to five-fold substantially improves the immune response in this segment of the population. Formulation of a “high-dose” vaccine for the elderly becomes a practical possibility with an unconstrained supply of antigen. A second set of advantages comes from eliminating the growth of virus from the manufacturing process. Currently, vaccine production strains are created by crossing the HA and NA genes from candidate circulating strains onto an egg-adapted virus, generally the PR/8/34 strain. Manufacturers then further adapt these production strains to create high-yield viruses. The adaptation process results in selection of mutations in the upper part of the HA globular head near the receptor binding site 
. As a consequence, the HA in the vaccine may be different from the HA in the original viral isolate. Prokaryotic expression, based on a cDNA copy of the original isolate HA, avoids genetic selection during the production process. In addition, there are situations where a suitable production strain cannot be created by conventional methods. In these cases, a related HA production strain is pressed into service. Eliminating the need to grow virus allows the production of the desired antigen with the original sequence.
In conclusion, the manufacturing approach described herein has major advantages over existing technologies in that it allows faster molecular development, rapid manufacturing and very high levels of productivity at small manufacturing scales. These advantages are critical to the successful production of seasonal and pandemic influenza vaccines.