The recent 2009-H1N1 swine-like virus influenza pandemic highlighted the need to rapidly produce enough vaccine doses for global vaccination brought to light the shortcomings of the traditional process of manufacturing influenza vaccines and the need to use alternative approaches for a more rapid generation of vaccine for global immunization in response to impending influenza pandemic. Bacterially expressed HA proteins can be manufactured rapidly and are amenable to mass production that can fulfill global vaccine needs. The main challenge to the prokaryotic production system is to ascertain proper refolding of expressed HA proteins representative of native HA spike structures on influenza virus. In addition to properly folded HA monomers, higher MW structures (i.e.,
trimers and oligomers) are important and likely to contribute to the optimal immunogenicity of the HA, since all influenza neutralizing antibodies are conformation dependent and some trimer specific antibodies have potent neutralizing activity 
. In eggs and mammalian cells, post-translational glycosylation contribute to the proper folding, trimerization and transport of the newly synthesized HA molecules to the cell membrane 
. However, in the case of recombinant HA proteins, trimerization is not always found even in eukaryotic cell substrates 
The main findings in the current study are: (a) bacterially expressed H1N1 HA1 (1–330) and HA (1–480) can be purified as properly folded proteins as determined by CD spectroscopy, SPR analyses with H1N1 immune sera, and adsorption of neutralizing activity from post-infection and post vaccination sera; (b) the HA1 (1–330) contained >50% trimeric and oligomeric forms and could bind to fetuin and agglutinated human RBC, while the HA (1–480) and mammalian cell expressed HA0 proteins were predominantly monomeric, did not bind fetuin, and did not agglutinate RBC; (c) the HA1 (1–330) induced higher titers of neutralizing antibodies compared with HA (1–480) or mammalian derived recombinant HA0 in rabbits and sheep; (d) in a ferret H1N1 challenge model, high-dose vaccination (30 μg HA) with both bacterially expressed HA1 (1–330) and HA (1–480) proteins protected animals from morbidity (elevated body temperature and weight loss) following challenge with novel H1N1 A/California/07/2009 virus. However, low-dose vaccination (7.5 μg HA) of ferrets with HA1 (1–330) resulted in lower morbidity and more rapid virus clearance compared with the HA (1–480) vaccinated group.
This study extends our previous reports with the H5N1 highly pathogenic virus, in which we have used whole-genome-phage display libraries (GFPDL) to map the antibody responses following human infection or vaccination. We have identified large HA1 fragments, encompassing the receptor binding domain (RBD), that were bound by broadly neutralizing human monoclonal antibodies from H5N1 recovered individuals and by their polyclonal convalescent sera 
. In a subsequent study, we found that following vaccination with inactivated H5N1 (A/Vietnam/1203/2004) influenza vaccine the immune sera from the MF59-adjuvanted vaccinated individuals bound with much higher avidity to bacterially expressed properly folded H5 HA1 proteins compared with unadjuvanted vaccine sera 
. Importantly, the bacterially expressed HA1 proteins were also shown to absorb most of the neutralizing activity in post infection and post vaccination sera 
. Based on these studies, we hypothesized that bacterially-expressed HA1 fragments if properly folded, could be useful as vaccines against emerging influenza strains.
In the current study, we found that expression and purification of properly folded H1N1 HA1 (1–330) (lacking HA2) in bacterial system was more efficient and gave higher yield compared with the larger HA ectodomain (1–480). Between 39–45 mg of >90% purified HA1 (1–330) protein can be obtained from 1 liter of bacterial batch culture, while the yield for HA (1–480) was only 0.4–0.8 mg/L. In addition to the much lower yield, the HA (1–480) contained only monomers, and as a result, it did not bind to fetuin and did not agglutinate RBC. Both of these functions require the presence of quartenary HA forms (i.e. trimers and oligomers). Mammalian expressed HA0 protein exhibited the same properties as the bacterially expressed HA(1–480) ectodomain. This is in agreement with previous reports on full length HA ectodomain proteins expressed in a variety of cell substrates wherein peptide linkers were introduced to facilitate oligomerization 
. Similar to our findings, the oligomerized cell-based HA product showed better neutralizing antibodies than its monomeric counterpart 
While both proteins were immunogenic in ferrets at the high dose of 30 μg, the HA1 (1–330) was more immunogenic and protected ferrets from H1N1 morbidity more efficiently at a lower dose (7.5 μg) compared with the HA (1–480) protein. In the case of mass vaccination, dose sparing is likely to be of great impact.
The ability of HA1 globular domain to form trimers has not been reported before. We have recently confirmed that bacterially expressed HA1 globular heads from multiple influenza strains (avian H5N1 and seasonal strains), can be produced at high yield. In all cases the HA1 proteins contain trimers and oligomers and agglutinate RBC. Studies to map the trimerization signal of HA1 are ongoing.
In the face of an impending influenza pandemic, HA1 proteins derived from the newly spreading virus can be rapidly expressed in bacterial systems several months before the traditional approach using vaccine strains generated via either gene reassortment or reverse genetics, followed by adaptation to growth in eggs. With appropriate testing methods in place to monitor proper folding and biological activity (hemagglutination assay), this simple and efficient approach may provide an early vaccine for large scale production to fulfill global vaccine needs in a much shorter time frame. Moreover, bacterially produced HA vaccines may also be an alternative for humans with known egg allergies that cannot be immunized with traditional influenza vaccines produced in eggs.