Figure 1.
Microneedle immunization with imiquimod-adjuvanted influenza subunit vaccine enhances IgG2a isotype responses.
Sera collected on days 14 and day 28 after immunization with adjuvanted influenza subunit vaccine were tested for virus-specific immunoglobulins by ELISA as described in materials and methods. Data are represented as arithmetic mean +/− standard deviation. Total IgG was detected by using HRP-conjugated goat anti-mouse IgG (A). IgG subclasses (IgG1 and IgG2a) were tested using sera collected on day 28 post-immunization. IgG subclasses were detected using HRP-conjugated goat anti-mouse IgG1 or IgG2a (B). (n = 9) *** - p<0.001.
Figure 2.
Imiquimod co-delivered with influenza subunit vaccine on coated microneedles induces higher HAI titers compared to vaccine alone.
Sera collected on day 28 after immunization were tested for HAI titers as described in materials and methods. HAI titers are reported as the reciprocal of the last dilution of serum inhibiting agglutination of red blood cells. Data represented as median value. (n = 9) *** - p<0.001.
Figure 3.
Microneedle immunization with imiquimod and influenza subunit vaccine elicits a robust IFN-γ cellular immune response.
Purified T cells from the spleen and total lymph node cells were stimulated for 4 days with 5 µg of influenza subunit vaccine supplemented with 30 U/mL of recombinant human IL-2. All data represented as arithmetic mean +/− standard deviation. IFN-γ and IL-4 were detected in cell culture supernatants by ELISA (A - purified T cells, B - draining lymph node). On day 4, stimulated cells from the draining lymph node were fixed and stained for surface markers and intracellular IFN-γ (C) and IL-4 (D). (n = 3) * - p<0.05, *** - p<0.001.
Figure 4.
Microneedle immunization with imiquimod and influenza subunit vaccine confers enhanced protection against lethal challenge with mouse adapted A/California/07/2009 virus.
Mice immunized with adjuvanted influenza subunit vaccine or vaccine alone were challenged with 5×LD50 (A) or 20×LD50 (B,C) of the mouse adapted A/California/07/2009 virus. (n = 6) * - p<0.05.
Figure 5.
Imiquimod induces improved clearance of replicating virus from the lung and reduced production of pro-inflammatory cytokines in response to challenge.
Mice immunized with adjuvanted influenza subunit vaccine or vaccine alone were challenged with 20×LD50 of mouse adapted A/California/07/2009 virus. All data are represented as arithmetic mean +/− standard deviation. Lungs were removed on day 4 post-challenge and processed for plaque assay (A) and TNFα (B) and IL-6 (C) cytokine ELISAs. (n = 3) * - p<0.05, ** - p<0.01.