The green platform for a new generation of vaccines

NELSON, G; MARZOL, E; LAGUÍA BECHER, M.; LÓPEZ, J.; PERIOLO, O.; LA TORRE, J.; MARCONI, P.; ALVAREZ, MA

Lisboa

Congreso; ICAR2012; 2012

Infectious diseases are challenged by using chemicals  (antimicrobials, non-antibiotic biocides), physical treatments, and also by strengthening the innate immune system (immunotherapy, immunomodulating agents, cytokines, etc.). Among this last category the developing of novel vaccines explores the use of different production platforms that could be advantageous both regarding the speed of vaccine production and the economy of the process. Considering the time needed to develop a production process the main concern is to have a platform that could give a fast response to face an epidemic outbreak. One of the attractive alternatives is the plant platform which has already demonstrated its capacity to express recombinant proteins, e.g.: antibodies and their fragments, enzymes, and also immunogens. We propose the use of a plant platform for expressing candidate antigens for a new generation of vaccines. In our laboratory we have demonstrated the efficiency of a plant platform based on  Nicotiana tabacum for expressing the glycoprotein E2, which is the main immunogen of the virus responsible of the Bovine Viral Diarrhoea (BVD), a disease broadly distributed in the world.  Specifically, we have expressed a truncated version of that protein (without its transmembrane domain); the construct also bears specific sequences for the successful expression of the protein in plants (the Arabidopsis 2S2 signal replacing the ss viral secretory signal peptide, the Kozak sequence, and the KDEL ER- retention signal). The promoter and  terminator used were from the Cauliflower Mosaic Virus (CaMV35S). The expression of the truncated version of E2 (tE2) was performed by infecting the N. tabacum leaves with an Agrobacterium tumefaciens strain bearing the designed construct. When the E2 glycoprotein was transiently expressed in tobacco leaves, the MW of the expressed protein was as expected (35 kDa), the highest yield obtained after 4 days of inoculation was 20  μg g-1 of infected leaf, corresponding to 1,3 % of total soluble protein content. The ability of developing the production of neutralizing antibodies was tested by vaccinating both guinea pigs (which is an accepted animal model for testing BVDV vaccines) and cattle with two formulations containing the plant extract and an oily (Montanide ISA 70 SEPPIC®) or an aqueous adjuvant (Al(OH)3). Animals were primed and boosted with 0.5 ml (guinea pigs) or 5.0 ml (cattle) of a vaccine formulated combining the adjuvant and an amount of plant extract containing 20 μg of antigen both in different relationships. The bleed sera of each animal were used for detecting neutralising antibodies by the Virus Neutralization Test (VNT) recommended by OIE and, the specific ELISA was performed for quantifying neutralizing antibodies anti-E2. High titers of antibodies were obtained from animals inoculated with the plant-made vaccine. In both cases the response was positive besides the typical individual heterogeneity of any animal model. Also, the sero-conversion obtained suggests a good potential performance for the recombinant antigen. The VNT demonstrated the induction of neutralising antibodies after inoculating with the plant recombinant tE2 vaccine