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Dengue virus (DENV) is an emerging threat causing an estimated 390 million infections per year. Dengvaxia, the only licensed vaccine, may not be adequately safe in young and seronegative patients; hence, development of a safer, more effective vaccine is of great public health interest. Virus-like particles (VLPs) are a safe and very efficient vaccine strategy, and DENV VLPs have been produced in various expression systems. Here, we describe the production of DENV VLPs in Nicotiana benthamiana using transient expression. The co-expression of DENV structural proteins (SP) and a truncated version of the non-structural proteins (NSPs), lacking NS5 that contains the RNA-dependent RNA polymerase, led to the assembly of DENV VLPs in plants. These VLPs were comparable in appearance and size to VLPs produced in mammalian cells. Contrary to data from other expression systems, expression of the protein complex prM-E was not successful, and strategies used in other expression systems to improve the VLP yield did not result in increased yields in plants but, rather, increased purification difficulties. Immunogenicity assays in BALB/c mice revealed that plant-made DENV1-SP + NSP VLPs led to a higher antibody response in mice compared with DENV-E domain III displayed inside bluetongue virus core-like particles and a DENV-E domain III subunit. These results are consistent with the idea that VLPs could be the optimal approach to creating candidate vaccines against enveloped viruses., (© 2020 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.