Inhibition of immunoglobulin E response to Japanese cedar pollen allergen (Cry j 1) in mice by DNA immunization: different outcomes dependent on the plasmid DNA inoculation method

Abstract

To develop a new immunotherapy for Japanese cedar (Cryptomeria japonica; CJ) pollinosis, we evaluated the use of DNA immunization by inoculating mice with plasmid DNA encoding Cry j 1 as a CJ pollen major allergen (pCACJ1). Repeated intramuscular (i.m.) inoculation of BALB/c mice with pCACJ1 produced anti-Cry j 1 antibody responses, which were predominately of the immunoglobulin G2a (IgG2a) type. Furthermore, this inoculation suppressed immunoglobulin E (IgE) and IgG1 antibody responses to subsequent alum-precipitated Cry j 1 injections. Splenic T cells isolated from mice inoculated with pCACJ1 i.m. secreted interferon-gamma (IFN-gamma), but not interleukin (IL)-4, in vitro upon stimulation with Cry j 1 as well as with p277-288, a peptide corresponding to the T-cell epitope of Cry j 1. In contrast, inoculation of BALB/c mice with pCACJ1 by gene gun injection caused response predominantly of the IgG1 type, and enhanced production of anti-Cry j 1 IgE antibodies to subsequent alum-precipitated Cry j 1 injections. Splenic T cells isolated from pCACJ1-innoculated mice by gene gun injection secreted both IFN-gamma and IL-4 in vitro, upon stimulation with Cry j 1 as well as with p277-288. These findings suggest that i.m. inoculation with pCACJ1 effectively elicits Cry j 1-specific T helper 1 (Th1)-type immune responses, resulting in inhibition of the IgE response to Cry j 1.

Figure 1

Western blot analysis of expressed Cry j 1. Lysates of the Cry j 1 transfectants and control were electrophoresed. Cry j 1 bands were detected using rabbit anti-Cry j 1 immunoglobulin G (IgG), as described in the Materials and methods. Lane 1, native Cry j 1; lane 2, lysate of pCACJ1-transfected 293-T cells; lane 3, lysate of pCAGGS-transfected 293-T cells. MW, molecular weight.

Figure 2

Intramuscular (i.m.) inoculation with pCACJ1 mainly induced immunoglobulin G2a (IgG2a) antibody response to Cry j 1. BALB/c mice (n = 4) were inoculated i.m. with 100 µg of pCACJ1 four times (4 × i.m.) at weekly intervals. The anti-Cry j 1 IgG, IgG1 and IgG2a titres of pooled sera from mice on days 12 (open bars), 24 (hatched bars) and 36 (solid bars) after the last inoculation were measured by enzyme-linked immunosorbent assay (ELISA). Data represent mean±SD of duplicate wells. The results are representative of three independent experiments. *IgG1 < 5·0 ng/ml.

Figure 3

Gene gun inoculation with pCACJ1 mainly induced antibody response of the immunoglobulin G1 (IgG1) type to Cry j 1. BALB/c mice (n = 4) were inoculated with 1 µg of pCACJ1 by gene gun twice (2 × g.g.) or three times (3 × g.g.) at weekly intervals. The anti-Cry j 1 IgG, IgG1 and IgG2a titres of pooled sera from mice on days 12 (open bars), 24 (hatched bars) and 36 (solid bars) after the last inoculation were measured by enzyme-linked immunosorbent assay (ELISA). The data are presented as mean±SD of duplicate wells. The results are representative of three independent experiments. *IgG2a < 5·0 ng/ml.

Figure 4

Intramuscular (i.m.) inoculation with pCACJ1 inhibited the immunoglobulin E (IgE) and immunoglobulin G1 (IgG1) responses to Cry j 1. BALB/c mice (n = 6) were inoculated i.m. with phosphate-buffered saline (PBS) or with 100 µg of pCAGGS or pCACJ1 four times at weekly intervals. Six weeks after the last inoculation, the mice were sensitized with 5 µg of Cry j 1 plus alum and given a booster of 1 µg of Cry j 1 plus alum 3 weeks after the sensitization. Anti-Cry j 1 IgE titre (a) of sera pooled from mice 7 days after the booster, or the anti-Cry j 1 IgG1 (b) and IgG2a (c) titres of sera pooled from mice on days 0 (open bars) and 14 (hatched bars) after the first sensitization, and on day 7 (solid bars) after the booster, were measured by enzyme-linked immunosorbent assay (ELISA). The data are presented as mean±SD of duplicate wells. The results are representative of three independent experiments. *IgG1 < 5·0 ng/ml; IgG2a < 5·0 ng/ml. PCA, passive cutaneous anaphylaxis.

Figure 5

Gene gun inoculation with pCACJ1 enhanced immunoglobulin E (IgE) and immunoglobulin G1 (IgG1) antibody responses to Cry j 1. BALB/c mice (n = 6) were or were not inoculated with 1 µg of pCAGGS or pCACJ1 by gene gun two or three times at weekly intervals. Six weeks after the last inoculation, mice were sensitized with 5 µg of Cry j 1 plus alum and given a booster of 1 µg of Cry j 1 plus alum 3 weeks after sensitization. The anti-Cry j 1 IgE titre (a) of pooled sera from mice 7 days after the booster, or the anti-Cry j 1 IgG1 (b) and IgG2a (c) titres of sera from six mice on days 0 (open bars) and 14 (hatched bars) after the first sensitization, and on day 7 (solid bars) after the booster, were measured by enzyme-linked immunosorbent assay (ELISA). The data are presented as mean±SD of duplicate wells. The results are representative of two independent experiments. *IgG1 < 5·0 ng/ml; IgG2a < 5·0 ng/ml. PCA, passive cutaneous anaphylaxis.