Recombinant immunogen rBm7462 expressed in a transgenic plant (Arabidopsis thaliana) as a new approach for...

The tick Rhipicephalus microplus causes significant economic losses, particularly in tropical and subtropical regions. The tick control research agenda has overlooked the recombinant expression of new-generation vaccines in plants. This study aims to produce the anti-tick immunogen rBm7462 (Bm86 epitopes) in the heterologous system of Arabidopsis thaliana for the first immunogenicity evaluation in BALB/c mice. We constructed a binary expression vector to generate recombinant clones by inserting a synthetic minigene previously designed to span the theoretical coding regions of the synthetic peptide SBm7462® in tandem repeats. To insert the vector, we used the Gateway® system to select the clones in a culture medium enriched with gentamicin (40 mg/L) and spectinomycin (100 mg/L). We selected transformant seedlings with the highest levels of recombinant peptide expression by generative selection. We performed RT-PCR, immunohistochemistry, and confocal microscopy to verify peptide expression in recombinant cells. We used SDS-PAGE and Western blotting techniques to verify the identity of the recombinant peptide. We evaluated the general immunogenicity features of the recombinant peptide expressed in the plant employing BALB/c mice, using orally administered leaves and a soluble protein extract subcutaneously at three time points. Posteriorly, we humanely euthanised these mice to obtain lymph nodes and the ileum for histopathological examination. The immunised groups of animals showed microscopic changes compatible with germinal centre (GC) formation and somatic hypermutation, with dendritic cell-like antigen expression detected by immunohistochemical staining in lymph nodes. We demonstrate that the recombinant peptide expressed in a transformed A. thaliana clone retains immunogenicity in mice, aligning with previous findings and opening a new avenue for the research agenda on tick vaccines.