Rapid high-level transient expression of Norwalk virus-like particles in Nicotiana benthamiana leaf and oral immunogenicity in mice

Norwalk virus (NV) is a member of the Caliciviridae family which causes epidemic gastroenteritis in the United States. Recent studies using new diagnostic assays have shown that the epidemiological significance of NV infections has been greatly underestimated. Mary Estes and colleagues have shown that NV contains a genome of single stranded positive sense RNA of about 7.6kb, which includes three open reading frames (ORFs). The second ORF encodes the Norwalk virus capsid protein (NVCP); 180 molecules of NVCP self assemble to form the 38 nm icosahedral viral capsid. Among the most promising viral antigen candidates for vaccine production are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing epitopes. The expression of the NVCP as vaccine antigen in potato tuber and tomato fruit and delivery via ingestion of transgenic material has already been proven successful, and oral delivery of plant tissue containing NVCP to human volunteers resulted in humoral and mucosal immune responses. In this work we used a tobacco mosaic virus (TMV) based viral vector replicating in Nicotiana benthamiana, to quickly produce large quantities of correctly assembled NV VLP for preclinical and clinical trials. High level of recombinant protein production, rapid expression, and scale up capabilities are the main advantages of this transient system. The proprietary viral expression technology was developed at Icon Genetics (Halle, Germany) and it is based on a deconstructed viral strategy that consists of in planta assembly of functional viral vectors from separate provector modules. The NVCP transiently expressed accumulated in the cytoplasm at levels up to 0.8 mg/g of fresh leaf material; it retains antigenic properties; and as demonstrated by sucrose gradient centrifugation, self assembles 38 nm virion size VLP, which co-sediment with those produced in insect cells. Such high expression level allows a more convenient smaller mass of plant material administered per dose by ingestion, making unit dosage of a human vaccine feasible in single gelatin capsule delivery systems. Results of preclinical studies using freeze dried leaf material for oral immunization of mice will be presented, and the utility of the rapid expression system discussed.