Covalent protein display on Hepatitis B core-like particles in plants through the in vivo use of the SpyTag/SpyCatcher system

Abstract

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.

Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana

“…During this study, a constant OD 600 of 0.4 for the agrobacteria infiltration suspension was used, as reported previously [ 48 ]. HBcAg construct and chimeric construct produced monomer, dimer, and tetramer forms of the recombinant proteins during the expression in N. benthamiana ( Figure 3 and Figure 4 , panel c), we also observed multimers above that, classic with plant-produced HBcAg constructs [ 49 ]. A two-step protocol was used for VLP purification [ 44 ].…”

Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts

“…The potential for steric hindrance must also be taken into account during enzyme immobilization. Complete occupancy was reported for tandem hepatitis B core VLPs attached to green fluorescent protein (GFP) or HIV capsid protein p24 in vivo, whereas a negative correlation between the size and density of displayed antigens was reported on Acinetobacter phage AP205 spherical VLPs (Thrane et al, 2016;Peyret et al, 2020). TMV is also able to build virus-like particles (VLPs).…”

“…TMV is also able to build virus-like particles (VLPs). But due to the physiological pH, this is not possible in planta as shown in Peyret et al (2020) for Hepatitis B core-like particles. For a successful assembly of TMV VLPs, the CP subunits have to be dialyzed against an acidic pH.…”

Microparticles and Nanoparticles from Plants—The Benefits of Bioencapsulation

“…If the tandem HBcAg core particles carry the SpyCatcher sequence (a 12.3-kDa portion of the CnaB2 domain), then any protein carrying the 13-amino-acid SpyTag can form an irreversible isopeptide bond. This has been used, for example, to conjugate the HIV antigen p24 to HBcAg VLPs in planta [ 136 ].…”

“…Since then, many other virus structural proteins have been expressed in plants, including foot and mouth disease coat protein and the HPV L1 coat protein [58,[120][121][122]. Following the successful production of VLPs based on HBsAg, more complex particles were produced containing more than one type of protein subunit, and even enveloped particles have now been expressed in plants [58,122,125,[134][135][136]. The hepatitis B core antigen (HbcAg) has been expressed not only as a potential VLP-based vaccine against hepatitis B virus but also as a carrier for other vaccines due to its strong inherent immunogenicity [134].…”