Figure 1.
Evaluation of NP specific IgG and IgG subclasses in serum.
Anti-NP IgG titers were determined by ELISA on day 60. BALB/c mice (n = 5) were vaccinated with two doses of rNP (10 µg/dose) alone or co-administered with c-di-AMP (10 µg/dose) by intranasal route, whereas control animals received PBS, at an interval of 3 weeks. (A) Anti-NP IgG titer. (B) Anti-NP IgG subclass titers. The results are expressed as mean end point titers. The S.E.M. is indicated by vertical lines. Differences were statistically significant at p<0.01 (**) or p<0.05 (*) with respect to values obtained in mice receiving the antigen alone.
Figure 2.
Evaluation of NP specific IgA in mucosal lavages.
NP-specific IgA titers measured by ELISA in nasal (NL) and BAL lavages of BALB/c mice immunized with two doses of rNP (10 µg/dose) alone or co-administered with c-di-AMP (10 µg/dose) by intranasal route at an interval of 3 weeks. Control animals received PBS. Results are expressed as NP specific IgA titer. The S.E.M. is indicated by vertical lines. Differences were statistically significant at p<0.001 (***) and p<0.01 (**), compared with the titers in mice immunized with rNP alone.
Figure 3.
Analysis NP-specific cellular responses induced by vaccination.
Splenocytes from immunized groups of mice were incubated for 24 or 48 h in the presence of rNP or peptides encompassing MHC class I and class II restricted epitopes of NP. The number of IFN-γ (A), IL-2 (B), IL-4 (C) and IL-17 (D) producing cells was then determined by ELISPOT. Results are expressed as number of spots of cells producing cytokines per 106 spleen cells after subtraction of background values of unstimulated cells. The differences are statistically significant p<0.001 (***) compared to the results of cells obtained from mice vaccinated with rNP alone.
Figure 4.
Analysis of proliferative responses in vaccinated mice.
Splenocytes from immunized groups of mice were re-stimulated for 96 h with different concentrations of rNP (A) or a pool of peptides encompassing a MHC class II restricted epitopes of NP. Cellular proliferation was measured by incorporation of [3H] thymidine into the DNA. The results are represented as stimulation index (SI).
Figure 5.
Protection of vaccinated mice against challenge with the influenza A virus.
BALB/c mice (n = 6) were immunized on day 0 and 21 with rNP (10 µg/dose) alone or co-administered with c-di-AMP (10 µg/dose) by intranasal route, whereas control animals received PBS. All groups were challenged on day 60 with a non-lethal dose of the influenza strain A/Puerto Rico/8/34 (H1N1). Animal body weight was then monitored for 14 days. Results are expressed as average percentage of weight loss.