Influenza virus remains an important respiratory pathogen with the potential to cause worldwide pandemics, such as the “Spanish” influenza virus in 1918 and the novel swine-origin H1N1 pandemic in 2009 
. Furthermore, the recent delays in the production of the monovalent H1N1 vaccine illustrate the need for a rapidly produced and safe alternative to egg-based influenza vaccines. Here we investigated the use of recombinant baculovirus expression to produce soluble forms of the HA protein, which is the primary target of neutralizing antibody. This system has several advantages including the high levels of protein expression 
and an established safety record in humans
. Current influenza vaccine production requires the generation of a high-yield egg growth reassortant to obtain a sufficient yield for vaccine production. Using molecular cloning, the HA gene from circulating viruses can be cloned directly from clinical isolates and a recombinant protein vaccine can be rapidly produced to prevent new seasonal outbreaks or pandemics as a result of antigenic drift or shift 
In this study, we generated a rBV-derived soluble hemagglutinin (sHA) modified at its C-terminus with the GCN4pII trimerization heptad repeat to stabilize the trimeric structure of the secreted protein. The heptad repeat is derived from the yeast transcription factor GCN4, which plays a role in the biosynthesis of amino acids during starvation. The wildtype heptad repeat forms stable dimers; however by mutating the ‘a’ and ‘d’ positions in the heptad repeat, trimeric or tetrameric coiled coils can form 
. This strategy has been used recently to study the structure and function of the HIV-1 envelope glycoprotein and parainfluenza virus 5 F protein 
The sHA selected for this study was derived from the H3N2 virus A/Aichi/2/68 due to the extensive data on its structure and function previously published 
. Our data indicated that the GCN4pII modification stabilized the trimeric sHA in solution as determined by BS3 crosslinking and analysis by Western blot. Mice primed and boosted with a low dose of recombinant sHA.GCN4pII without an adjuvant had an improved humoral response to the H3N2 HA compared to mice receiving the unmodified sHA protein. Our results are also consistent with results using the H3N2 virus A/Victoria/3/75 soluble HA in which the monomeric HA induced HA specific antibodies; however there was no detectable binding to the trimeric viral HA
. In addition, mice vaccinated with the A/Victoria/3/75 sHA were not protected against challenge with homologous mouse adapted virus.
Other studies using recombinant influenza sHA have used HA from H1 
, H3 
, H5 
, and H7 
subtypes in addition to influenza B hemagglutinin 
as a vaccine antigen. The properties of these sHA have been analyzed using hemagglutination assay, trypsin susceptibility, and/or electron microscopy 
. Overall, the ability of these recombinant proteins to form stable trimers varies among subtypes and viruses. In the current study, we have employed a water-soluble crosslinker, BS3, which links monomers within an oligomeric structure to characterize the trimeric structure of the recombinant H3 sHA 
. Crosslinking provides the advantage of being able to determine the approximate ratio of oligomers in solution without the need to use complex size-exclusion chromatography techniques. Modification of the H5 HA with the foldon trimerization domain from the T4 fibritin protein also was reported to generate soluble trimeric HA in the recombinant baculovirus system which was more immunogenic than the unmodified protein when mice were vaccinated with recombinant protein with adjuvant 
. In addition, the GCN4 trimerization repeat has been used to study the immunogenicity of H5 HA in poultry
. Our data clearly demonstrate that the enhanced immunogenicity of the trimeric H3 HA is due to the presentation of native trimeric epitopes in the GCN4pII stabilized protein, while the unmodified protein presents epitopes associated with the altered low-pH conformation of the HA protein.
Our data indicate that sHA.GCN4pII generates higher levels of both IgG2a and IgG1 than the soluble Aichi HA. The ratio of IgG1:IgG2a that we observe following boosting suggests that the cytokine environment is a balanced Th1/Th2 phenotype in the sHA.GCN4pII group while the mice immunized with sHA exhibit a Th2 phenotype. In the influenza mouse model, virus specific class 1 helper T cells (Th1) have been suggested to play a more important role in viral lung clearance compared to class 2 helper T cells (Th2) which are associated with increased airway inflammation and enhanced morbidity
. Furthermore, the higher serum levels of IgG2a, which is regulated by Th1-associated cytokines
, in sHA.GCN4pII vaccinated mice might suggest that the finding of more efficient clearance of virus could be related to the effector functions of IgG2a such as complement activation and Fc receptor binding
. Therefore, both the quantity and quality of the humoral immune response in addition to the helper T cell phenotype should be considered in evaluating influenza vaccine efficacy.
It is noteworthy that the protection observed did not require the use of any adjuvants in the vaccination, although we cannot exclude any undocumented adjuvant properties of the GCN4pII repeat. Using the modified recombinant HA, we were able to induce complete protection in mice following a lethal challenge with mouse adapted A/Aichi/2/68 after two vaccinations. Furthermore, we were able to provide protection to 5/6 of mice following a single vaccination with 3 µg of sHA.GCN4pII in the absence of adjuvants. Thus, the trimeric antigen has a high potential to induce protective immune responses with just a single vaccination, which may be further enhanced by including approved adjuvants.
These results demonstrate that using the expression construct employed in this study, the instability of HA is eliminated by stabilizing the trimeric structure through the GCN4pII trimerization heptad repeat. Furthermore, the stabilized trimeric HA has been demonstrated to exhibit enhanced immunogenicity over unmodified HA with just one vaccination in the absence of adjuvants. These differences could be explained by the presence of the low-pH conformation in the unmodified sHA detected by monoclonal antibody binding. Previous studies on the structure of the X31 HA (A/Aichi2/68) monomer indicate that the trimeric protein is dissociated into monomeric subunits
. Antibodies that recognize this low pH structure might interact with epitopes at the monomer-monomer interface, or the “silent face”, of the hemagglutinin molecule. By presenting the “silent face” in the unmodified sHA, the antibody repertoire maybe skewed to epitopes not accessible in live virus at neutral pH. Therefore, by conserving the native trimeric structure of the HA, the sHA.GCN4pII represents a better influenza subunit vaccine candidate.