One of the key findings from our previous study was that HA antigens from H1 and H3 serotypes had different structure preferences in order to elicit optimal protective antibodies [11] . Two of the HA antigen designs used in that study were also included in the current study to identify the optimal design for the H5 serotype HA antigens: one used the wild type HA antigen insert (H5.wt), which has the exact same amino acid sequences found in the natural viral isolate, and the other used a truncated HA antigen insert (H5.dTM), which removed the transmembrane (TM) and intracellular segments of the HA2 domain ( Fig. 1) . In addition, a third HA antigen insert was created (H5.tPA), in which a human tissue plasminogen activator (tPA) sequence replaced the original wild type leader sequence from the HA antigen (Fig. 1) . The third HA antigen design was adopted because the H5.dTM design also used a tPA leader sequence and the H5.tPA insert served as a control for the H5.dTM insert to understand the role of the tPA leader when it is incorporated as the only change in the design from the original wild type HA antigen insert.