Studies were conducted in this report to identify the mechanism responsible for better protective antibodies in rabbit immune sera elicited by the H5.tPA HA insert design but the exact mechanism is currently unclear. First, we tested whether a higher level of HA antigen expression was produced with the tPA-leader design. As shown in Fig. 2A and 3A, antigen expression levels between WTleader design and tPA-leader design were similar, thus excluding this possibility. Next, we asked whether there is increased secretion of the HA antigen due to the use of the tPA leader. However, as shown in Fig. 2A and 3A , there is no major detectable level of secreted HA antigens in supernatant for either the WT-design or tPA-design. Furthermore, the dTM design did have a higher level of secretion due to the deletion of transmembrane and intracellular portion of HA protein but did not elicit better protective antibody responses. Finally, a study was conducted to examine the possible role of post-translational processing, such as a change in glycosylation, of HA the antigen, which may affect the antigen processing pathway, as we previously reported with a hepatitis B surface antigen DNA vaccine [33, 34, 35] . However, Figure S1 showed that there is no major difference between WTdesign and tPA-design after de-glycosylation treatment.