Immunogenicity of recombinant E2 protein of bovine viral diarrhea virus expressed in transgenic alfalfa plants

MARIA SOL PEREZ-AGUIRREBURUALDE; CRISTINA GOMEZ; ANDREA PECORA; ARDILA F; ANDRES WIGDOROVITZ; DUS SANTOS MARIA JOSE

Congreso; XXVIII World Buiatrics Congress; 2014

Bovine viral diarrhea virus (BVDV) is a pestivirus of worldwide distribution and is a major cause of economic losses in livestock. The main control strategies are vaccination and segregation of persistently infected animals. As prevention tool subunit vaccines offer many advantages, however, the challenge is to generate a protective immune response at an affordable cost for veterinary applications. Previously, our group reported the development of a subunit experimental vaccine based on transgenic alfalfa plants expressing a truncated version of the E2 glycoprotein of BVDV without the transmembrane domain and fused to a single chain antibody against MHCII (APCH-tE2). In order to assess the immunogenic properties of the vaccine 18 (n = 6 each group) female pregnant cows were randomly divided into 3 groups. One group was immunized with 2 doses of APCH 3ug-tE2, with an interval of 30 days between doses. Following the same design, the positive control group was immunized with an inactivated virus vaccine with viral titer of 107 TDIC 50/ml (BVDV-1a) and the negative control group with a negative extract of a non transgenic alfalfa plant. All groups were boosted one year after first immunization in order to evaluate the anamnesic response evoked by the vaccines following a regular vaccination schedule. Serum samples and PBMC s were collected at various time points for the measurement of BVDV-antibodies and cellular immune response. Also, after birth calves were sampled to evaluate the transference of passive immunity through colostrums. The humoral response was assessed by serum neutralization test against homologous and heterologous virus and by blocking and avidity ELISA assays. To assess the cellular immune response, mononuclear cells were purified by density gradient Ficoll-Paque Plus to proceed with LPDA (antigen-dependent lymphoproliferation) against the purified tE2 protein expressed in mammalian cells. Cell proliferation was evaluated using the CFSE staining to measure the proliferating cells and to further detect T cell subsets by flow cytometry. The experimental trial involving animals was approved by the Institutional Committee for the Care and Use of Experimental Animals of INTA (CICUAE). Subunit vaccine induced neutralizing antibody titers against the homologous virus vaccine strain higher than 3 (evaluated by Reed-Muench´s method) being similar to those generated by the whole virus vaccine. Antibody levels remained high until day 240 post vaccination and were still detectable at day 360. After the booster, neutralizing antibody titers quickly reached values higher than 3. The antibodies curves after vaccination showed a similar dynamic of increased avidity in both, BVDV and APCH-tE2 vaccinated groups. As expected, APCH-tE2 became a marker vaccine since it allowed differentiating vaccinated animals from infected ones. The evaluation of calves? serum samples showed an efficient transference of humoral immunity from the cows vaccinated with the experimental vaccine, during the first two month of life. In concern to the cellular response T cell activation showed similar levels of proliferation, comparable to the control group after booster. A year later of primovaccination when the animals received the second booster, proliferating T cell subsets were detected (CD4+/CD25+ and CD8+), demonstrating efficient priming of the lymphocyte population by the antigen expressed in transgenic plants. In conclusion, these results along with the efficiency afforded by the vaccine a