3.3 pCIgD-Man-L increased specific BoHV-1 antibodies titers in cattle
Cattle received vaccination at 0, 30 and 60 days. When antibody kinetics was evaluated, pCIgD and pCIgD-Man-L were able to induce specific anti-BoHV-1 antibody levels (Fig.3a ). However, significant differences in pCIgD-Man-L at 60 dpv (p<0.05) were found compared with pCIgD group. After challenge assays (90 dpv) all animals seroconverted (data not shown).
In addition, ELISA isotype titer at 90 dpv against BoHV-1 was analyzed (Fig. 3b ). Interestingly, there were significant differences in IgG1 (p<0.001) for the pCIgD and pCIgD-Man-L group versus pCIneo, but in IgG2 difference was observed only for pCIgD-Man-L (p<0.05) with respect to the pCIneo group.
No significant differences among groups were found when neutralizing antibodies were measured; however, a trend of increasing neutralizing antibodies was observed in pCIgD-Man-L over pCIgD treated groups (data not shown).
Antibodies in nasal swabs were also evaluated. It is reported that the presence of antibody in nasal mucosa confer protection against respiratory viral penetration. Here, IgA isotype anti-BoHV-1 level in nasal swabs at 6 days post challenge (dpc) was evaluated (Fig. 3c ). The results showed that the IgA titer in swabs of the pCIgD-Man-L treated group was significantly different compared with pCIneo treated group (p<0.05).
pCIgD and Man-L induce Dendritic Cells Activation in vitro
Dendritic cells are the major antigen presenting cell (APC) and are key initiators of antiviral responses (Ludewig et al., 2000). They endocitose, process and present antigens to naive T cells and act as messengers between the adaptive and innate immune responses. Afferent lymph dendritic cells (ALDCs) from bovine were used to elucidate if DNA alone or with loaded targeted liposomes were able to positively modulate dendritic cells. ALDCs were incubated in vitro  with vaccines pCIgD, pCIgD-Man-L (with or without LPS from Brucella ovis ) or Man-L alone (with or without LPS from Brucella ovis ). CD40 surface molecules were significantly upregulated with respect to mock for pCIgD (p<0.01), pCIgD-Man-L with (p<0.05) and without LPS (p<0.01). Also, CD40 was upregulated positively when treated with Man-L with LPS (p<0.001) but not with Man-L without LPS. These results indicate that pCIgD alone and liposomes including LPS can activate dendritic cells (Fig.4) .