Stable expression of the E2 protein from the bovine viral diarrhea virus in in vitro cultures from tobacco and the humoral immune response induced in cows by tobacco-agroinfiltrated leaf extracts

Abstract

Our goal was to compare transient y stable plant expression strategies for the production of the E2 protein from Bovine Viral Diarrhea Virus (BVDV) y to test the ability to induce the production of neutralizing anti-E2 antibodies in cattle from an experimental vaccine containing agroinfiltrated tobacco leaf extracts expressing the E2-ER version. Stably transformed tobacco calluses y cell suspensions expressing the apoplast-directed (E2-Apo) y endoplasmic reticulum-retained (E2-ER) E2 protein versions have been established. The E2 expression levels in calluses were similar for both versions y were estimated to be 3 µg/g fresh weight (FW), corresponding to 1.15% of total soluble protein (TSP). The E2-Apo y E2-ER proteins were detected in the biomass of cell suspension cultures but only after sample concentration, indicating that calluses produce higher levels of E2 than cell suspension cultures. On the other hand, immunization of cattle with an experimental vaccine constituted by an aqueous extract containing E2 transiently expressed in tobacco leaves (60 µg of E2-ER) y Al(OH)₃ Hydrogel adjuvant (90:10) produced a high level of anti-E2 antibodies (87.5 % seroconversion) with titers considered to be immunoprotective. The E2 expression levels in calluses were significantly higher than those obtained in cell suspension cultures but lower than those attained by transient expression in agroinfiltrated leaves. The immunized cattle developed a strong humoral response showing the potential of the plant-made E2 protein to develop a subunit vaccine against BVDB.