*3.1. Serum IgG and IgA Antibody Responses*

The anti-influenza serum IgG and IgA titers in NMRI mice were significantly higher than baseline in animals receiving WIV combined with N3 adjuvant or N3 combined with FliC-DNA adjuvant already at day 21 after a single immunization (Figure 1A,C).

**Figure 1.** Influenza A H1N1/WIV/SI specific serum IgG and IgA titers. NMRI mice (**A**) and C57BL/6 mice (**B**) IgG titers 3 weeks (day 21) after one immunization and 4 weeks (day 90) after the booster immunization shown for each group. Each bar shows geometric mean (GMT) serum titer, and error-bars show 95% confidence interval values for each study group. Influenza A H1N1 specific serum IgA titers in NMRI mice (**C**) and C57BL/6 mice (**D**) three weeks (day 21) after one immunization and four weeks (day 90) after the booster immunization shown for each group. Bars show geometric mean (GMT) serum titer, and error-bars show 95% confidence interval values for each study group. Significant differences are indicated by nonparametric Mann–Whitney U analysis p-values. n.s. = not significant.

WIV given with pFliC(-gly) DNA did not result in significantly higher serum IgG titers then seen in mice that were immunized with only WIV demonstrating the important contribution of N3 to the effect of pFliC(-gly). The highest serum IgG titers were seen when both N3 and pFliC-DNA were combined with WIV with 100–140-fold increased titers. Booster immunizations at day 60 resulted in further elevation of the IgG and IgA titers (Figure 1A,C). Three weeks after booster immunization, the serum IgG in non-adjuvanted WIV immunized animals increased two-fold and IgA-titers increased five-fold. However, with WIV immunizations containing N3, a 100-fold serum IgG and 50-fold IgA increase was seen over WIV alone. Similar increases were also observed in mice receiving WIV with N3 and pFliC(-gly). Thus, the humoral influenza-specific immune responses between mice receiving WIV with N3 or N3 and FliC-DNA were not significantly different when analyzed by ELISA. Thus, binding antibodies alone may provide a misleading immune pattern from a functional point of view, as can be seen in the more detailed assays, such as subclass IgG ELISA (Figures 2–4) or functional antiviral assays, such as in virus-inhibition assays (Figure 5). IgG isotype comparisons revealed a correlation with the use of pFliC-DNA and the appearance of stronger IgG2a responses (Table 2).

**Figure 2.** The anti-influenza A/H1N1/SI whole inactivated viral (WIV) specific serum IgG1 and IgG2a/2c subclass reactivity OD490 reactivity in serum collected at 21 days post primary immunization and 30 days post booster immunization (day 90). The median and range OD490 reactivity is shown for each study group (n = 6–8 animals/group), illustrating the results that are shown in Table 2. Significance. \* = *p* ≤ 0.05, \*\* = *p* ≤ 0.01, \*\*\* = *p* ≤ 0.001.

**Figure 3.** Influenza A H1N1 specific lung wash IgA and IgG. IgA anti-WIV titer in NMRI and C57BL/6 mice four weeks after booster immunization (day 90) (**A**). IgG anti-WIV titer in NMRI and C57BL/6 mice four weeks after booster immunization (day 90) (**B**). Titers presented are as anti-WIV titer/mg total IgA or IgG. Bars show GMT, and error-bars show 95% confidence intervals for each study group. Significant differences are indicated by Mann–Whitney U analysis p-values.

**Figure 4.** Anti-influenza A/H1N1/2009pdm rHA in lung-wash subclass IgG1 and IgG2a/IgG2c ELISA reactivity seen in three study groups at day 90 post primary immunization. (**A**) show IgG1 and IgG2a median subclass ELISA reactivity (and range) in lungs wash samples collected from outbread NMRI mice. The value given on top of each pair of bars indicates the median IgG1/IgG2a ratio in the group. (**B**) show IgG1 and IgG2c median subclass ELISA reactivity (and range) in lungs wash samples collected from inbread C57BL/6 mice. The value given on top of each pair of bars indicates the median IgG1/IgG2c ratio in the group. \* = *p* ≤ 0.05, \*\* = *p* ≤ 0.01, \*\*\* = *p* ≤ 0.001.

**Figure 5.** Influenza A H1N1/SI specific serum HAI titers. Titers in NMRI mice (**A**) three weeks after one immunization (day 21) and 4 weeks after the booster immunization (day 90) for each group of mice. Titers in C57BL/6 mice (**B**) three weeks after one immunization and four weeks after the booster immunization for each group of mice. Bars show GMT serum titer, and error-bars show 95% confidence intervals for each study group. Significant differences are indicated by nonparametric Mann–Whitney U analysis *p*-values.

The most pronounced effect on the IgG1/IgG2a ratio was seen after the primary immunization with WIV in animals receiving N3 with pFliC-DNA (*p* < 0.01) in contrast to WIV alone or WIV with only N3 or pFliC-DNA alone. Both the IgG1 and IgG2a serum titers increased after the booster immunization, but the IgG1 titers increased more than IgG2a titers. In all groups of influenza vaccine immunized mice receiving WIV with all adjuvants both subclasses IgG1 and IgG2a responses were seen, which indicated a mixed Th1/Th2 immune response. Nevertheless, the inclusion of pFliC(-gly) skewed the response away from Th2.

In C57BL/6 mice, significant serum IgG and IgA titer increase over baseline was only seen when the influenza vaccine and N3 adjuvant was used alone or when N3 was combined with pFliC(-gly) (Figure 1B,D). Similar to NMRI mice, when WIV was given with N3 or N3 and pFliC(-gly), a 10-fold and 20-40-fold increased serum IgG was observed, respectively. As observed in the NMRI mice, WIV given with pFliC(-gly) DNA did not result in significantly higher serum IgG titers than that seen in mice that were only immunized with WIV. After a booster-immunization serum, the IgG titers were doubled and IgA titers increased four-fold in vaccinated non-adjuvanted C57BL/6 mice. However, mice receiving booster WIV immunization with N3 increased the serum IgG titers 100-fold and 1000-fold when N3 was combined with pFliC(-gly) as compared to WIV alone. When compared to mice receiving WIV alone, serum IgA titers increased 20 to 100-fold with the use of N3 or N3 with pFliC(-gly). Similar to NMRI mice, C57BL/6 mice receiving WIV and pFliC(-gly) immunizations did not have significant increases in antigen-specific IgG or IgA responses at either day 21 or 90. IgG isotype comparisons revealed a higher Th1-like response in mice that were given WIV with N3 and pFliC-DNA (Table 2). Booster immunization induced a mixed Th1/Th2-type immune response with increases in the titer of both IgG subclasses. As with the NMRI mice, after the booster immunization both the IgG1 and IgG2c serum titers increased, but the IgG1 titers increased more than the IgG2c titers. The inclusion of pFliC(-gly) also skewed responses to a Th1-like IgG isotype.
