Modulation of Antigen Display on PapMV Nanoparticles Influences Its Immunogenicity

Abstract

Background: The papaya mosaic virus (PapMV) vaccine platform is a rod-shaped nanoparticle made of the recombinant PapMV coat protein (CP) self-assembled around a noncoding single-stranded RNA (ssRNA) template. The PapMV nanoparticle induces innate immunity through stimulation of the Toll-like receptors (TLR) 7 and 8. The display of the vaccine antigen at the surface of the nanoparticle, associated with the co-stimulation signal via TLR7/8, ensures a strong stimulation of the immune response, which is ideal for the development of candidate vaccines. In this study, we assess the impact of where the peptide antigen is fused, whether at the surface or at the extremities of the nanoparticles, on the immune response directed to that antigen. Methods: Two different peptides from influenza A virus were used as model antigens. The conserved M2e peptide, derived from the matrix protein 2 was chosen as the B-cell epitope, and a peptide derived from the nucleocapsid was chosen as the cytotoxic T lymphocytes (CTL) epitope. These peptides were coupled at two different positions on the PapMV CP, the N- (PapMV-N) or the C-terminus (PapMV-C), using the transpeptidase activity of Sortase A (SrtA). The immune responses, both humoral and CD8+ T-cell-mediated, directed to the peptide antigens in the two different fusion contexts were analyzed and compared. The impact of coupling density at the surface of the nanoparticle was also investigated. Conclusions: The results demonstrate that coupling of the peptide antigens at the N-terminus (PapMV-N) of the PapMV CP led to an enhanced immune response to the coupled peptide antigens as compared to coupling to the C-terminus. The difference between the two vaccine platforms is linked to the enhanced capacity of the PapMV-N vaccine platform to stimulate TLR7/8. We also demonstrated that the strength of the immune response increases with the density of coupling at the surface of the nanoparticles.

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants

“…Since the production platform we describe here does not require the resulting VLPs to be infectious, it may be possible to modify the particles more extensively than previously possible by modifying either the coat protein subunits, incorporating designer RNAs, or both. For example, PapMV, expressed in Escherichia coli and assembled on ssRNA in vitro, has been studied recently for its potential use as vaccine epitope carrier and adjuvant [ 32 , 33 , 34 ]. The use of plants as a production system may have benefits over bacterial expression, especially in the case of the display of peptides which may benefit from expression in a eukaryotic host.…”

Convenient Auto-Processing Vector Based on Bamboo Mosaic Virus for Presentation of Antigens Through Enzymatic Coupling

“…It has been reported that the coupling density of M2e peptide onto the PapMV-N nanoparticles (made of PapMV CP with N-terminal acceptor site) could reach up to 83% by increasing the incubation time and the concentration of M2e peptide ( 22 ). However, in our system, the efficiency of SrtA-mediated ligation was only around 20% to 40% ( Figures 3C, F and 4D ).…”

“…In contrast to the previous designs, our current system exhibits several unique features. Firstly, the BaMV CVP scaffolds are produced in plants, as opposed to those produced in bacteria or animal cells ( 16 – 18 , 21 , 22 , 56 , 57 ). The plant production system reduced the risk of contamination from prokaryotic or animal sources, and allows for easy scale-up.…”

“…Secondly, our design facilitated auto-processing of CP and self-assembly CVP scaffold. In comparison, the scheme for PapMV-based platform recently published ( 22 ) requires pH and temperature shift with buffer exchange and an incubation time of 16-24 hrs for intein removal following the harvesting of PapMV CP from the bacterial cells. Thirdly, our system provides a vector for the expression of target antigens with improved solubility by fusing with the 6×His-CaM-6×His tag, which could be removed upon ligation with the B5G CP by SrtA.…”

“…It has been shown that different SrtA may recognize and cleave at specific amino acid sequences in the donor protein and mediate the ligation of the acceptor protein at the exposed N-terminus ( 19 , 20 ). The coupling of target proteins on the surfaces of VLPs or CVPs by SrtA-mediated ligation has been successfully applied in different systems, including bacterial phage M13 ( 21 ) and papaya mosaic virus (PapMV) ( 17 , 18 , 22 ). However, these platforms could be further improved with regard to the convenience in the mass-production of VLP or CVP scaffolds, which is another major concern in the development of VLP or CVP antigen-presentation platforms.…”