Engineering, production, characterization of G protein variants in Nicotiana benthamiana and animal immunogenicity studies

Rabies is a viral disease caused by the rabies virus (Lyssavirus) that affects the central nervous system, ultimately leading to death and brain defects. Rabies usually transmitted through a bite among domestic dogs and wild carnivorous animals. The rabies virus (RABV) infects the central nervous system causing disease in the brain and death. Rabies is 100% fatal if untreated and ultimately causing more than 70,000 deaths annually worldwide, especially among children in developing countries. Rabies can be prevented by vaccination and can be cured immediately after infection. Although several human and animal vaccines against rabies are available, they are expensive, have relatively low immunogenicity and also difficult to produce. Therefore, less expensive, more immunogenic and safe rabies vaccines that can be produced in the large quantities are urgently needed. The transient plant expression system has become a more attractive and promising platform for the expression of a wide range of recombinant proteins such as vaccines, antibodies, therapeutic proteins, including mammalian complex proteins. In this study, we engineered and produced truncated forms of glycoprotein (G protein) variants, namly RG2 (monomoeric) and RG3 (trimeric) of rabies virus in Nicotiana benthamiana plant for the first time, using transient expression system. Plant produced RG2 and RG proteins were purified through Ni-NTA column chromatography. The purification yields of RG2 was about ~32, the purification yield of RG3 was ~20 mg/ kg plant leaf. The results of immunogenicity studies showed that the plant produced G-protein variants elicited significantly high antibody titers in mice. Mice immunized with 5 g of RG3 elicted higher titer antibody compared to that of RG2 immunized with the same amount of protein. Plant-produced G-protein variants could be a cost-effective, safe and highly immunogenic rabies vaccine candidate.