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Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants

Bovine viral diarrhea virus (BVDV) is considered an important cause of economic loss within bovine herds worldwide. In Argentina, only the use of inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. The use of recombinant subunit vaccines has been proposed as an alternative to overcome this difficulty. Different studies on protection against BVDV infection have focused the E2 protein, supporting its putative use in subunit vaccines. Utilization of transgenic plants expressing recombinant antigens for the formulation of experimental vaccines represents an innovative and cost effective alternative to the classical fermentation systems. The aim of this work was to develop transgenic alfalfa plants (Medicago sativa, L.) expressing a truncated version of the structural protein E2 from BVDV fused to a molecule named APCH, that target to antigen presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa leaves was 1ug/gr at fresh weight and its expression remained stable after vegetative propagation. A methodology based an aqueous two phases system was standardized for concentration and partial purification of APCH-tE2 from alfalfa. Guinea pigs parentally immunized with leaf extracts developed high titers of neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated with 3 ug of APCH-tE2 produced in alfalfa transgenic plants showed complete virological protection.

Expression of a ScFv-E2T fusion protein in CHO-K1 cells and alfalfa transgenic plants for the selective targeting to antigen presenting cells

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an important cause of mortality, morbidity and economical losses of cattle with a worldwide distribution. Subunit vaccines provide the opportunity to develop safe vaccines. However, the challenge is to generate a protective immune response to a cost affordable for veterinary applications. Here we provide two strategies to overcome this problem: vaccine immunogenicity augmentation and immune response manipulation, by antigen targeting to MHC class II molecules on antigen presenting cells (APCs), and cost reduction, by antigen production in alfalfa transgenic plants. As part of the first strategy, a truncated version of the E2 glycoprotein (E2T) from BVDV, without its transmembrane domain, was fused (or not) to a single chain antibody fragment (ScFv), recognizing a conserved region of MHC class II molecules, and stably expressed in mammalian cells (CHO-K1). Proteins were purified by affinity chromatography (IMAC) from cell culture supernatants, and quantified by SDS-PAGE and Coomasie blue staining. Stable cell line productivity and protein purity were around 0.5 mg/L and 85 %, respectively. Targeting resulted in an increase in antigen surface-binding and internalization by APCs, as evidenced by flow cytometry analysis. It also provoked a concomitant augmentation of T cell stimulation and proliferation in vitro. Furthermore, antigen targeting resulted in a polarization shift from a Th1-type immune response to a Th2-type. The non-targeted vaccine induced IFN-γ expression, whereas the targeted vaccine induced predominantly IL-4, as revealed by qPCR. Bovine animals immunized with the targeted vaccine induced an adaptive immune response and neutralizing antibody production, at lower doses than the non-targeted vaccine (2 immunizations with a 0.2 ug dose). As part of the second strategy, alfalfa transgenic plants were generated by infection of petioles with recombinant Agrobacterium tumefaciens, which contained the appropriate genes cloned in a pCAR vector downstream of CsVMV (Cassava vein mosaic virus) promoter. Transgenic plants expressed recombinant proteins at levels around 1 ug/g fresh leaf. Vaccines formulated with plant extracts were capable of inducing an immune response and neutralizing antibody production in cattle, using a schedule of 3 immunizations with a 1 ug dose. In consequence, both strategies were successful for developing more efficient vaccines against BVDV, and demonstrate the benefits and potential from a rational design of vaccines.

8th International Veterinary Immunology Symposium (8thIVIS)

My work "Expression of a ScFv-E2T fusion protein in CHO-K1 cells and alfalfa transgenic plants for the selective directioning to antigen presenting cells", presented at the 8th International Veterinary Immunology Symposium (8thIVIS), held in Ouro Preto (Brazil) in 2007, was awarded as the second best student poster.