<br><br />Materials and methods<br /><br><br />Results<br /><br><br />Discussion<br />Secreted HA is a favorable antigen for swine influenza vaccines as the additional processing steps required to extract and purify membrane-associated HA from insect cells, as has been described for human vaccines, can become cost prohibitive for veterinary products (Cox et al., 2008). As oligomeric HA has been shown to be more immunogenic than monomer HA, secreted forms of HA require modifications to potentially oligomerize or increase their immunogenicity by other means such as increasing antigen half-life or enhancing uptake by antigen presenting cells (Zhang et al., 2009; Weldon et <img src="http://farm5.static.flickr.com/4336/37211209291_1e0f21c013.jpg
" align="right" width="211" style="padding:10px;"/> al., 2010). Similar to reports of previous work in mouse models, our study demonstrates that HA fused to an IgG Fc domain elicits robust HI antibody titers in pigs and provides protection against challenge with a heterologous H1α cluster SIV (Loureiro et al., 2011; Zaharatos et al., 2011; Du et al., 2011). Interestingly, we found that the HA-2aFc vaccine generated significantly increased HI titers and provided pigs with protection comparable to that elicited by the baculovirus-displayed and FeLV gag VLP-displayed HA vaccines. These results are surprising given that VLP-based influenza vaccines have been reported to provide superior protection against influenza strains in mice when compared with purified recombinant HA vaccines (Bright et al., 2008). As the H1-BD and H1-Fgag vaccines were formulated using hemagglutination titers and the H1-2aFc vaccine was formulated based on gel densitometry measurements, the HA concentrations were different between the particle-based vaccines and the H1-2aFc vaccine. Although an accurate determination of HA content could not be established using gel densitometry methods for the H1-BD and H1-Fgag antigens, given their unpurified state, SDS-PAGE evaluation of all <a href='http://www.angiotensin-iii-human-mouse.com/forum.php?mod=viewthread&tid=2620&extra=
'>three calcium calmodulin dependent protein kinase </a> suggested that the H1-2aFc vaccine contained a greater HA input (data not shown). Given the increased HA content of the H1-2aFc vaccine it’s difficult to compare this antigen presentation to the particle-based presentations in terms of antigen quality (i.e. availability of immunologically relevant epitopes that elicit HI antibodies). Although the increased HI titers produced by the H1-2aFc vaccine were most likely due to the increased HA content of the vaccine, a future evaluation of these antigens using equal HA input levels may reveal which presentation of HA generates the most robust HI antibody response in pigs.<br />Despite comparable protective efficacy in pigs, the baculovirus-displayed HA and FeLV gag VLP-displayed HA vaccines may be a more feasible option for SIV vaccine development. In addition to the increased HA content of the H1-2aFc vaccine, purification by protein A affinity chromatography was required for the H1-2aFc antigen as a means to concentrate the protein due to low yields from the BacDB-H1-2aFc-infected SF+ cells. Despite reported yields of 2–5mg/L of a H4-human Fc fusion protein using the BEVS, the process reported in this work would require further optimization for commercial applications as yields were in the hundreds of micrograms per liter range, necessitating large volumes of harvest material to produce sufficient antigen for the trial (Loureiro et al., 2011). In contrast to the H1-2aFc antigen, the baculovirus-displayed and VLP-displayed HA antigens evaluated in this study were utilized directly from harvest supernatant and not purified via sucrose gradient centrifugation as has been previously described for multiple studies (Pushko et al., 2005; Prabakaran et al., 2008; Chen et al., 2010; Tang et al., 2010; Pyo et al., 2012; Tretyakova et al., 2013). The alleviation of the need for further processing of these particle-based methods allows for more streamlined and cost-efficient production processes that are vital for veterinary vaccines.<br />As a single placebo group vaccinated with adjuvanted media was incorporated in the study, further consideration should be given to the potential influence of the IgG2a Fc, baculoviruses or FeLV gag VLPs on the outcome of the study. From previously published reports, it may be possible that these moieties could elicit an adjuvant effect, inducing a more robust immune response than with HA alone (Hervas-Stubbs et al., 2007; Loureiro et al., 2011). It is uncertain, but unlikely that vaccination with IgG2a Fc or FeLV gag VLPs alone results in a protective response in pigs. It has been reported that non-inactivated baculovirus can induce non-specific protection of mice from lethal challenge with H1N1 influenza (Abe et al., 2003). The reported protection was a result of robust innate immune responses and occurred only when 1.1×108 PFU wild-type baculovirus was administered intranasally 24h prior to challenge. When mice were vaccinated subcutaneously or intramuscularly with wild-type baculovirus, no protection was observed. We would expect that non-specific protection of pigs against H1N1 SIV from intramuscular vaccination with IgG Fc alone or FeLV gag alone would follow a similar model. As the pigs in this study were immunized twice intramuscularly and challenged 14days after the second vaccination, the contribution of the swine innate responses, as was described previously for mice, are expected to be negligible. In addition, SIV H1-specific humoral responses were detected in the serum of pigs immunized with any of the vaccines further confirming the specificity of the elicited immune response. Still, the potential for cross-reacting <a href='http://www.biocompare.com/Antibodies/
'>antibodies</a> elicited by the IgG2a Fc or FeLV gag has not been evaluated and is worthy of further investigation in future studies.