Abstract: Objective To 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. Methods The 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. Results The 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. Conclusion We have successfully constructed a highly immunogenic
pcDNA3.1-Hum DNAvaccine that can mediate immune protection by inducing Foxp3+Treg cells.