An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

Virus like particles (VLPs) are a very promising subunit vaccine candidate. VLPs derived from viruses that infect the gastrointestinal system like the Norwalk virus (NV) are very well suited to study mucosal immunization and oral delivery of antigens. Stable chromosomal plant transformation has been extensively and successfully investigated for the production and delivery of recombinant NV like particles (rNV). The major disadvantages of this approach are the long generation time necessary to establish the transgenic lines and the low level of transgene expression. Here we report the high and rapid production, ~0.8mg/g of fresh weight (FW) in 12 days post infection (dpi), of the sNVCP by using an efficient Tobacco mosaic virus (TMV) derived expression system in leaves of N. benthamiana. Evaluation by sucrose gradient and electron microscopy showed proper assembly into rNV. Moreover we describe the strong systemic and mucosal immune response, upon oral delivery, of two different doses, 100 µg and 250 µg respectively, of partially purified plant derived rNVs administered to CD1 mice with and without 10 µg of cholera toxin (CT) as mucosal adjuvant. The rNV specific serum IgG and mucosal IgA were evaluated by enzyme-linked immunosorbent assay, kinetics of the production of different types and subtypes of immunoglobulins are reported. These results demonstrate the feasibility and advantages of using plant viral transient systems to produce an effective rNVs based mucosal vaccine in N. benthamiana.