Immunostimulant patch enhances immune responses to influenza virus vaccine in aged mice

Abstract

Improvement in the immune response to influenza virus vaccination in the elderly represents the primary unmet need in influenza virus vaccination. We have shown that topical application of immunostimulating (IS) patches containing heat-labile enterotoxin of Escherichia coli (LT) enhances immune responses to injected vaccines. We extend these findings and show that LT-IS patch application enhances the antibody responses to influenza virus vaccination in both young and aged mice. LT-IS patches markedly increased influenza virus-specific immunoglobulin G (IgG), hemagglutination inhibition antibody, mucosal antibody, and T-cell responses. The magnitude of the immune responses in aged mice receiving an LT-IS patch was equivalent to or greater than that of the immune responses in young mice given vaccine alone. These results suggest that addition of an LT-IS patch may compensate for the deficient immune function seen in the aged in response to influenza virus vaccination. Therefore, use of an LT-IS patch could be a new, safe, and simple immunization strategy that may significantly improve the outcome of influenza virus vaccination in the elderly.

FIG. 1.

A single immunization with an LT-IS patch significantly improves the responses to injected influenza virus vaccine. Young (∼2 months old) and aged (18 months old) C57BL/6 mice were immunized with 5 μg of trivalent split influenza virus vaccine (1.7 μg each of the A/New Caledonia, A/Panama, and B/Johannesburg strains) by i.d. injection. Patches containing 25 μl of PBS (0 μg of LT) or 10 or 50 μg of LT were applied to the skin over the injection site immediately after parenteral injection for 18 h. All mice were immunized on study day 0, and serum was collected 2 weeks postimmunization (day 14). Titers of IgG to A/New Caledonia (A and D), A/Panama (B and E), and B/Johannesburg (C and F) in serum were determined by an ELISA method. Individual titers are represented by open circles (young mice) or closed circles (aged mice). The geometric mean of each group is indicated by a horizontal bar. Prebleeding samples had titers of <80 ELISA units for young mice and <230 ELISA units for aged mice.

FIG. 2.

LT-IS patch enhances secondary responses to influenza virus vaccine delivered by i.m. injection. Young (∼2 months old) and aged (18 months old) C57BL/6 mice were immunized i.m. on days 0, 14, and 28 with 5 μg of trivalent split influenza virus vaccine as indicated in the legend to Fig. 1. Serum was collected, and titers of antibodies to the three strains of influenza virus vaccine were determined by ELISA. Results are reported as described in the legend to Fig. 1.

FIG. 3.

LT-IS patch enhances secondary responses to influenza virus vaccine delivered by i.d. injection. Young (∼2 months old) and aged (18 months old) C57BL/6 mice were immunized i.d. on days 0, 14, and 28 with 5 μg of trivalent split influenza virus vaccine as indicated in the legend to Fig. 1. Serum was collected, and titers of antibodies to the three strains of influenza virus vaccine were determined by ELISA. Results are reported as described in the legend to Fig. 1.

FIG. 4.

LT antibody titers are reduced in aged mice compared to responses in young mice. Young and aged C57BL/6 mice were immunized on days 0, 14, and 28 with 5 μg of trivalent split influenza virus vaccine by i.m. (A and B) or i.d. (C and D) injection. Patches containing PBS or LT (10 or 50 μg) were applied over the injection site for ∼18 h. Serum was collected, and titers of antibody to LT were determined by ELISA. Results are reported as described in the legend to Fig. 1. Prebleed samples had titers of <50 ELISA units.

FIG. 5.

Mucosal responses to influenza virus B/Johannesburg are significantly enhanced by an LT-IS patch. Young and aged mice were immunized as described in the legend to Fig. 1, with trivalent influenza virus vaccine by i.m. injection. A patch containing PBS (0 μg) or 10 μg of LT was applied over the site of the i.m. injection. Lung washes were collected 3 weeks after the third immunization and analyzed for B/Johannesburg-specific IgG (A to D) and IgA (E to H) by ELISA. Young and aged naive mouse lung washes had nearly undetectable levels of IgG and IgA (OD, <0.1).

FIG. 6.

Mucosal responses to influenza virus A/Panama are significantly enhanced by an LT-IS patch. Young and aged mice were immunized as described in the legend to Fig. 5. Lung washes were collected 3 weeks after the third immunization and analyzed for A/Panama-specific IgG (A to D) and IgA (E to H) by ELISA. Young and aged naive mouse lung washes had nearly undetectable levels of IgG and IgA (OD, <0.1).

FIG. 7.

LT-IS patch increases the number of IL-4-producing cells. Young and aged mice were immunized as described in the legend to Fig. 1. Three weeks after the third immunization, single-cell suspensions were prepared from the spleens, inguinal lymph nodes (LN), and lungs and measured in an ELISPOT assay for the frequency of IL-4-secreting cells when cultured in the presence of CM or CM containing 10 μg of influenza virus A/Panama (Flu A) or influenza virus B/Johannesburg (Flu B) per ml. The number of IL-4-producing cells per 10⁶ cells was plotted.