Recombinant protein technology may provide a rapid response to emerging influenza infections with pandemic potential. The current vaccine campaign is based on a traditional process that depends on the derivation of reassortant viruses in mammalian cells followed by adaptation to high growth in eggs, which may result in mutations in the key antigenic sites. In addition, vaccine potency reagents require repeat vaccinations of sheep with bromelain cleaved HA from egg-grown vaccine strains. Therefore, recombinant technology can alleviate some of the bottlenecks and assist in production of early vaccines or potency reagents for vaccine lot release. Transient expression or random integration based cell lines are widely used for protein expression, but these technologies have several significant shortcomings including time-consuming, unpredictable and unreproducible levels of gene expression primarily due to lack of control over the position of integration. To that end, it will be important to use a certified mammalian cell line with a built in genomic site specific recombination cloning site and an optimized expression vector that can be used within short time frame for cloning and selection of stable integrants secreting properly folded HA proteins 
In the current study we provide the first data supportive of the use of 293 Flp-In based stable mammalian system for influenza hemagglutinin expression. The main findings were (a) Both HA1 and HA0 proteins from H5N1 HP A/Vietnam/1203/2004 were successfully expressed in 293 Flp-in cells and stable integrants were selected within 10-14 days; (b) the secreted HA1 and HA0 were purified from the supernatants of cells growing in serum free conditions and were shown to be properly folded as determined by binding to conformation dependent human MAbs; (c) the HA0 with intact polybasic cleavage site contained cleaved (HA1 and HA2) as well as uncleaved molecules (similar to HA on virions); (d) rabbit vaccination with the HA1 and HA0 proteins resulted in homologous virus-neutralizing antibodies with a significant cross neutralization of heterologous strains representing recently transmitted H5N1 clades; and (e) sera from humans vaccinated three times with the licensed H5N1 A/Vietnam/1203/2004 inactivated vaccine had lower titers against the homologous virus and negligible neutralizing antibody titers against heterologous H5N1 strains from multiple clades.
The use of site-directed recombinant technology for HA production in stable mammalian expression cell lines eliminates the handling of live highly pathogenic H5N1 viruses. It also circumvents the need for generation of egg-adapted high growth virus in the face of impending pandemic. High-level protein expressing mammalian cell lines could be reproducibly constructed based on the exchange of HA expression cassettes in a pre-tagged desired locus of the engineered cell line by site-specific recombination.
Our data supports and extend the findings of Wei et al. 
and Bosch et al. 
who used transient expression systems for expression of HA proteins. In this study, both secreted HA1 and HA0 expressed in the Flp-In system in the same integration site and were purified from the culture supernatants. The proteins were properly folded and were recognized by conformation dependent H5N1 neutralizing human MAbs.
Following vaccination, the kinetics of HA-binding antibody development (measured by SPR) paralleled the appearance of potent broadly neutralizing antibodies confirming the immunogenicity of Flp-in derived secreted HA1 and HA0 proteins. From the rabbit studies, the Flp-In derived HA1 (1-330) protein seems as immunogenic as intact HA0 (1-500) in generation of broadly H5N1 neutralizing antibodies and might be a promising vaccine candidate.
Importantly, the neutralization titers and breadth of cross clade neutralization observed in the current study using recombinant HA proteins, were higher than the neutralization titers measured in the sera of human volunteers vaccinated with escalating doses of the currently licensed H5N1 inactivated vaccine. Suguitan et al.
previously demonstrated that restoring the polybasic cleavage site in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, suggesting that removal of polybasic cleavage site due to safety concerns, had a deleterious effect on the immunogenicity and efficacy of the H5N1 HA 
. Therefore, it is plausible that the HA0 with intact polybasic cleavage site expressed in Flp-In system, which was cleaved into HA1 and HA2 forms provided better immunogenicity than what has been earlier reported for HA with mutated cleavage site 
In the face of impending pandemic, generation of vaccine in a timely manner is critical. While adjuvants were shown to improve the immunogenicity of inactivated vaccines against H5N1 and SOIV-H1N1 
, there is still a considerable time gap between virus isolation and generation of reassorted vaccine seed, as was evident during the recent SOIV-H1N1 pandemic. Therefore, recombinant technologies may provide an important early vaccine response alternative. The Flp-In system provides an advantage over the transient transfection approach.
Based on these data it will be prudent to derive a 293 Flp-In system that can be used for production of HA proteins for vaccine generation as well as reagents that can be used for quality assurance and vaccine release purposes. In addition to human vaccines, poultry vaccination may become an important early approach for curtailing the spread of newly emerging HP viruses and their transmission to susceptible human populations