Feasibility of Cowpea chlorotic mottle virus-like particles as scaffold for epitope presentations

Abstract

Background & MethodsWithin the last decade Virus-Like Particles (VLPs) have increasingly received attention from scientists for their use as a carrier of (peptide) molecules or as scaffold to present epitopes for use in subunit vaccines. To test the feasibility of Cowpea chlorotic mottle virus (CCMV) particles as a scaffold for epitope presentation and identify sites for epitope fusion or insertion that would not interfere with virus-like-particle formation, chimeric CCMV coat protein (CP) gene constructs were engineered, followed by expression in E. coli and assessment of VLP formation. Various constructs were made encoding a 6x-His-tag, or selected epitopes from Influenza A virus [IAV] (M2e, HA) or Foot and Mouth Disease Virus [FMDV] (VP1 and 2C). The epitopes were either inserted 1) in predicted exposed loop structures of the CCMV CP protein, 2) fused to the amino- (N) or carboxyl-terminal (C) ends, or 3) to a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.ResultsHigh levels of insoluble protein expression, relative to proteins from the entire cell lysate, were obtained for CCMV CP and all chimeric derivatives. A straightforward protocol was used that, without the use of purification columns, successfully enabled CCMV CP protein solubilization, reassembly and subsequent collection of CCMV CP VLPs. While insertions of His-tag or M2e (7-23 aa) into the predicted external loop structures did abolish VLP formation, high yields of VLPs were obtained with all fusions of His-tag or various epitopes (13- 27 aa) from IAV and FMDV at the N- or C-terminal ends of CCMV CP or N∆24-CP. VLPs derived from CCMV CP still encapsulated RNA, while those from CCMV CP-chimera containing a negatively charged N-terminal domain had lost this ability. The usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines was demonstrated with the synthesis of chimeric CCMV VLPs containing selected sequences from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus at both termini of the CP protein.ConclusionsCCMV VLPs can be successfully exploited as scaffold for epitope fusions up to 31 aa at the N- and C-terminus, and at a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.

Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus‐like particles

“…Scale bars: 200 nm. While CPs of numerous viruses accommodate genetic fusions and retain their ability to self-assemble into VLPs, those are commonly restricted to peptides as exemplified with simian virus 40 (Takahashi et al, 2008), bacteriophage P22 (Servid et al, 2013) or CCMV (Brumfield et al, 2004;Hassani-Mehraban et al, 2015) rather than full-length proteins. Another common limitation observed with VLPs is linked to sites of insertion of foreign proteins not necessarily located at the extremities but often exposed within loops of the CP, as it is the case for hepatitis B virus (Kratz et al, 1999;Peyret et al, 2015) or CPMV (Porta et al, 2003), requiring the inserted proteins to be fused to both their extremities.…”

Production and characterization of virus-like particles of grapevine fanleaf virus presenting L2 epitope of human papillomavirus minor capsid protein

“…VLPs from plant viruses have been produced in various microbial expression systems, namely Escherichia coli , Pseudomonas fluorescens , Pichia pastoris and Saccharomyces cerevisiae [26, 31–34]. These expression systems include prokaryotic and eukaryotic organisms, each having its own advantages and drawbacks.…”

“…However, the lack of post-translational modifications and the presence of endotoxins are the most intriguing problems when using these systems [35]. Moreover, since large amount of proteins is often produced in an insoluble form, extra denaturation and refolding processes may be required, as it was the case for CCMV [26]. Concerning the yeast expression system, the quantity of the produced proteins is usually lower than in E. coli .…”

Quantum dot encapsulation in virus-like particles with tuneable structural properties and low toxicity

“…for targeting) conjugated to outer surface while its inner cavity can host different types of cargo. 36 Furthermore, unlike adenoviruses, CCMV and other plantderived viruses are non-pathogenic for humans and are regarded as safe for potential human use 37 and have a broad in vivo biodistribution without any toxicity. 38 Therefore, CCMV-based protein cages hold a great promise as biocompatible nanoscaffolds for applications in nanomedicine such as targeted drug delivery systems.…”