Transient Plant Expression of Recombinant Rabies Virus Glycoprotein: A strategy for Vaccine Production

The rabies virus causes neurological infections, resulting in the deaths of over 60,000 people worldwide each year. Currently available rabies vaccines are attenuated virus vaccines, which carry the risk of transmission among both humans and animals. An exciting alternative to these traditional vaccines is the use of recombinant plant-based vaccines. In this study, we applied the rabies virus glycoprotein (RVGP) as a recombinant vaccine to be transiently expressed in plants. To achieve this, we fused human immunoglobulin G Fc with the KDEL sequence, a motif for endoplasmic reticulum (ER) retention (FcK), to the RVGP, to generate RVGP-FcK. We then cloned the RVGP- FcK gene expression cassette into the pEAQ vector and agro-infiltrated Agrobacteriumtumefaciens (LBA4404) carrying the pEAQ RVGP-FcK vector into the leaves of Nicotiana benthamiana for transient expression. A RT-PCR analysis confirmed the transcription of the RVGP-FcK gene, which was evident as early as four days post-infiltration (dpi). To optimize the spatial and temporal aspects of RVGP-FcK production, we conducted analyses of its expression levels at different leaf positions (top, middle, and base) and dpi. A western blot analysis demonstrated that the RVGP-FcK protein reached its highest expression level at 7 dpi in the top leaf position and at 5 dpi in the middle leaf position. However, no detectable expression was observed in the bottom leaves at any time point. Subsequently, we validated the functionality of RVGP-FcK through an ELISA analysis. The results revealed that RVGP-FcK was expressed and assembled into its functional form most effectively at 5 and 7 dpi in the top leaf position and at 5 and 7 dpi in the middle leaf position. Our findings demonstrate that the transient expression of a functional RVGP-FcK protein can be optimized spatially and temporally in plants.