Inactivation and purification of cowpea mosaic virus-like particles displaying peptide antigens from Bacillus anthracis

Abstract

Chimeric cowpea mosaic virus (CPMV) particles displaying foreign peptide antigens on the particle surface are suitable for development of peptide-based vaccines. However, commonly used PEG precipitation-based purification methods are not sufficient for production of high quality vaccine candidates because they do not allow for separation of chimeric particles from cleaved contaminating species. Moreover, the purified particles remain infectious to plants. To advance the CPMV technology further, it is necessary to develop efficient and scalable purification strategies and preferably eliminate the infectivity of chimeric viruses. CPMV was engineered to display a 25 amino acid peptide derived from the Bacillus anthracis protective antigen on the surface loop of the large coat protein subunit. The engineered virus was propagated in cowpea plants and assembled into chimeric virus particles displaying 60 copies of the peptide on the surface. An effective inactivation method was developed to produce non-infectious chimeric CPMV virus-like particles (VLPs). Uncleaved VLPs were separated from the contaminating cleaved forms by anion exchange chromatography. The yield of purified chimeric VLPs was 0.3 g kg(-1) of leaf tissue. The results demonstrate the ability to generate multi-gram quantities of non-infectious, chimeric CPMV VLPs in plants for use in the development of peptide-based vaccines.

Virus‐based nanoparticles as platform technologies for modern vaccines

“…Three major approaches are used to insert additional peptides into the virus coat protein, resulting in a coat protein fusion or chimera: direct fusion, linker fusion, and the “protein overcoat” strategy. In the direct fusion approach, the foreign peptide is linked directly to the N-terminus 26–28 or C-terminus 28–31 of the coat protein or inserted into flexible surface loops presented on the capsid surface 29, 32 . Although the external surface is usually chosen for presentation of native antigens recognized by B cells, the internal surface may be more suitable in some applications that involve the presentation of processed peptides 33, 34 .…”

Peptide‐Controlled Access to the Interior Surface of Empty Virus Nanoparticles

“…[5] In preparations of virus particles from infections the majority of particles contain RNA and are thus infectious unless they are subjected to UV, [6] chemical [7] or pH treatment. [8] However, such treatments can perturb the structural properties of the particles, and, with the exception of the last, do not actually remove the RNA, so the particles cannot be loaded with heterologous material.…”

Virus-Based Nanoparticles as Versatile Nanomachines

“…Procedures have therefore been established to render them noninfectious, including the heterologous expression of genome-free VLPs (157) and in vitro disassembly and reassembly to yield VLPs from self-assembled purified coat proteins (11, 158). Alternatively, nucleic acids can be removed from intact particles by alkaline hydrolysis (159, 160) either during extraction or following the recovery of pure virus particles (161). Shortwave (254 nm) UV irradiation can be used to cross-link and inactivate the genomes of intact particles (162).…”