OBJECTIVETo construct a humulus pollen allergy DNA vaccine pcDNA3.1-Hum and investigate its effect for immune protection mediated by Foxp3(+)Treg cells in asthmatic mice.

METHODSThe target humulus gene obtained from pTripIEx2-Hum plasmid by double enzyme digestion was inserted sequentially into pcDNA3.1(-) vector to generate the recombinant plasmid pcDNA3.1-Hum, which was validated by sequencing. The pcDNA3.1-Hum plasmid was transfected into COS-7 cells and the expression of the ectopic protein was analyzed using Western blotting. Co-cultured dendritic cells and CD4(+)CD25(-) T cells were stimulated with the expressed protein to test its efficacy in inducing Foxp3(+)Treg cells. The levels of humulus-specific IgE and IgG2a were assayed to evaluate the allergenicity and immunogenicity of pcDNA3.1-Hum in mice. The immunoprotective effect of pcDNA3.1-Hum was assessed in a mouse model of humulus-specific asthma.

RESULTSThe constructed pcDNA3.1-Hum plasmid was validated by sequencing and Western blotting, and the expressed protein was shown to induce Foxp3(+)Treg cells in the co-culture. In normal mice, pcDNA3.1-Hum induced a significant increase of humulus-specific IgG2a but had no effect on IgE. In the asthmatic mice, pcDNA3.1-Hum significantly decreased inflammatory cell counts and eosinophil percentages in the BALF, ameliorated lung inflammation, and lowered AHR and IL-4 levels; immunization of the mice with pcDNA3.1-Hum reversed humulus-induced reduction of serum IFN-γ and prevented the humulus-triggered reduction of Foxp3(+)Treg cell percentage in the spleen.

CONCLUSIONWe have successfully constructed a highly immunogenic pcDNA3.1-Hum DNA vaccine that can mediate immune protection by inducing Foxp3(+)Treg cells.