Expression of Rabies Glycoprotein and Ricin Toxin B Chain (RGP–RTB) Fusion Protein in Tomato Hairy Roots: A Step Towards Oral Vaccination for Rabies

Abstract

Transgenic hairy roots of Solanum lycopersicum were engineered to express a recombinant protein containing a fusion of rabies glycoprotein and ricin toxin B chain (rgp-rtxB) antigen under the control of constitutive CaMV35S promoter. Asialofetuin-mediated direct ELISA of transgenic hairy root extracts was performed using polyclonal anti-rabies antibodies (Ab1) and epitope-specific peptidal anti-RGP (Ab2) antibodies which confirmed the expression of functionally viable RGP-RTB fusion protein. Direct ELISA based on asialofetuin-binding activity was used to screen crude protein extracts from five transgenic hairy root lines. Expressions of RGP-RTB fusion protein in different tomato hairy root lines varied between 1.4 and 8 µg in per gram of tissue. Immunoblotting assay of RGP-RTB fusion protein from these lines showed a protein band on monomeric size of ~84 kDa after denaturation. Tomato hairy root line H03 showed highest level of RGP-RTB protein expression (1.14 %) and was used further in bench-top bioreactor for the optimization of scale-up process to produce large quantity of recombinant protein. Partially purified RGP-RTB fusion protein was able to induce the immune response in BALB/c mice after intra-mucosal immunization. In the present investigation, we have not only successfully scaled up the hairy root culture but also established the utility of this system to produce vaccine antigen which subsequently will reduce the total production cost for implementing rabies vaccination programs in developing nations. This study in a way aims to provide consolidated base for low-cost preparation of improved oral vaccine against rabies.

Plant‐made oral vaccines against human infectious diseases—Are we there yet?

“…The expression level of the RGP‐RTB fusion protein in different tomato hairy root lines ranged from 1.4 to 8 μg/g of tissue. A partially purified RGP‐RTB fusion protein was able to induce an immune response in BALB/c mice after intramucosal immunization, but the IgG titres were low (Singh et al ., ).…”

Production of vaccines for treatment of infectious diseases by transgenic plants

“…However its in vivo activity and immunogenicity have not been tested. Recently, Singh et al (2015) expressed rabies virus glycoprotein fused to ricin toxin B chain in tomato hairy roots. Fusion gene construct contained Nterminal ER-trageting Calreticuline signal sequence from tobacco and C-terminal ERretention signal SEKDEL and was under control of CaMV 35S double enhancer promoter.…”

Current Developments and Future Prospects for Plant-Made Biopharmaceuticals Against Rabies

“…Moreover, biomass propagation can be optimized and production times reduced [43,44]. An study conducted by Singh et al [45] shows results of engineered tomato hairy roots to achieve the expression of a fusion protein comprising the G protein and the ricin toxin B chain (RGP-RTB). The latter component is intended to serve as a highly effective vaccine adjuvant as the ricin B chain sugar-binding moieties interact with terminal galactosides in the cell membrane.…”

“…The process resulted in 200 g/l of biomass, with a recombinant protein content of up to 8 lg/g fresh weight tissue. In terms of immunogenic activity, partially purified RGP-RTB fusion protein induced humoral immune responses in mice when administered orally without accessory adjuvants [45].…”