*3.3. Hemagglutination Inhibition*

Although increases in serum HAI titers were observed at day 21 when comparing WIV to WIV and adjuvant groups, significantly increased HAI titers were only detectable after booster immunizations (Figure 5).

Booster immunization of both mouse strains with WIV with N3 or N3 and pFliC(-gly) significantly raised their HAI titer against A/H1N1/SI virus between four- and 32-fold. In mice receiving WIV immunization alone or WIV with pFliC(-gly), at best a non-significant doubling of HAI titer was seen. However, when combined with N3, pFliC(-gly) was able to promote a significant increase in HAI over WIV with N3 alone. None of the tested mice from any of the groups showed HAI titers against the A/H3N2/Wuhan strain. In general, the C57BL/6 mice developed lower serum HAI titers. These results indicate that the immune responses that were elicited by WIV together combined with N3 and N3 with pFliC(-gly) adjuvants were able to elicit clear increases in HAI titer that were well above the benchmark level of ≥40. Among the NMRI mice, all of the influenza immunized groups had animals that developed HAI-serum titer of 40 or more. The most significant responses were seen in groups where N3 and N3 combined with FliC-DNA was used as adjuvants, where all animals/group developed HAI antibody titers and the highest serum titers reached 65,000 at day 90. Among C57BL/6 mice, only animals in the two groups where N3 adjuvant was used with influenza antigen responded with HAI titers over 40. The highest HAI titers were seen in the group receiving N3 and FliC-DNA as adjuvant, with the highest HAI titers of 2560 being obtained at day 90 post-immunization.

## *3.4. Interleukin-5 Release Responses*

A significantly higher amount of Interleukin-5 (IL-5) secretion was produced from animals immunized with WIV combined with adjuvants when the spleen cells at day 90 were stimulated in vitro with WIV Influenza A virus (A/H1N1/SI) (Figure 6).

The highest average amounts were observed in NMRI mice (Figure 6A) in WIV with N3 adjuvant as compared to WIV alone. The addition of pFliC(-gly) to WIV led to lower IL-5 production, however this difference was not significant. However, NMRI mice receiving WIV and N3 with pFliC(-gly) had a significantly lower secretion of IL-5 production after influenza antigen restimulation. In naïve mice only given adjuvant, no IL-5 secretion was seen when stimulated with WIV. Restimulated spleen cells from C57BL/6 mice produced, on average, one-third of the IL-5 amounts that were observed in NMRI mice (Figure 6B). Cells from all mice immunized with WIV and adjuvant produced significantly higher IL-5 amounts than WIV alone immunized mice. However, no significant difference in IL-5 production was observed between the WIV and N3 vaccinated mice and those that were given WIV and pFliC(-gly) or WIV with N3 and pFliC(-gly). Together, these results demonstrate that WIV immunization with N3 leads to cellular immune responses that were capable of IL-5 production, which can be attenuated by the addition of pFliC(-gly).

**Figure 6.** Splenocyte IL-5 release in response to Influenza A recall. (**A**) Influenza A specific IL-5 secretion (median pg/mL) million spleen cells at 48 h in NMRI mice, four weeks after booster immunization. (**B**) Influenza A specific IL-5 secretion (median pg/mL) million spleen cells at 72 h in C57BL/6 mice, four weeks after booster immunization. Block figures show median culture medium IL-5 concentrations after in vitro stimulation with WIV A/H1N1/SI, and error-bars show maximum and minimum values for each study group. The different time points chosen for the two mouse strains were determined in an in vitro pre-study influenza/ConA stimulation of spleen cells, and the optimal time point for highest levels of IL-5 secretion in spleen cells of adjuvant-vaccinated aged animals was chosen. Significant differences are indicated by nonparametric Mann–Whitney U test *p*-values.
