Versatile RHDV virus‐like particles: Incorporation of antigens by genetic modification and chemical conjugation

Abstract

Virus-like particles have proved to be excellent molecular scaffolds, yet the individual characteristics and immune responses generated against each VLP requires the development of a wide range of capsids for use as vaccines, molecular delivery vessels, and nanoscale templates. Here we describe the development of Rabbit haemorrhagic disease virus (RHDV)-like particles as a rapidly versatile molecular workbench, overcoming limitations imposed by established genetic antigen incorporation procedures with chimeric VLP. Production of the RHDV capsid protein in a baculovirus system led to the self-assembly of VLP which were recovered at over 99% purity and manipulated both genetically and chemically. Fusion of small peptide sequences to RHDV VLP was well tolerated, forming chimeric capsids that enhanced the presentation of foreign peptide to hybridoma T helper cells 700-fold. Rapid and simple conjugation techniques employing the hetero-bifunctional chemical linker sulfo-SMCC enabled both small peptides and whole proteins to be conjugated to the surface of RHDV VLP, overcoming limitations imposed on VLP formation and yield experienced with chimeric VLP. Administration of VLP/ovalbumin conjugate provoked high titre ovalbumin-specific antibody in mice, demonstrating the immune stimulatory properties of the capsid were conferred to conjugated foreign antigen. VLP facilitated delivery of conjugated antigen to dendritic cells, eliciting proliferative responses in naïve TCR transgenic T helper cells that were at least 10-fold greater than ovalbumin antigen delivered alone.

High-Resolution Cryo-Electron Microscopy Structures of Murine Norovirus 1 and Rabbit Hemorrhagic Disease Virus Reveal Marked Flexibility in the Receptor Binding Domains

“…Three liters of cell culture yielded 0.5 to 1.0 mg of purified virus with a particle/PFU ratio of less than 100. Baculovirus expression and purification of recombinant RHDV (rRHDV) VLPs were performed as previously described (8). Cryo-electron microscopy (EM) data were collected at the National Resource for Automated Molecular Microscopy (NRAMM) facility in San Diego, CA (4).…”

Virus-Like Particles, a Versatile Subunit Vaccine Platform

“…Each VLP is unique, with optimal expression identifi ed through trial and error by comparing the translated products of multiple expression systems. For example, Rabbit Haemorrhagic Disease Virus (RHDV) VLPs can be optimally produced by expressing the RHDV VP60 capsid protein in Spodoptera frugiperda (SF) cells using a recombinant baculovirus vector (Young et al 2004(Young et al , 2006Peacey et al 2007 ). Icosahedral T = 3 VLPs with a diameter of around 40 nm spontaneously form when VP60 is expressed in SF21 cells.…”

Functional Virus-Like Particles Production Using Silkworm and Their Application in Life Science

“…This chimera gag protein forms HIV-like particles in insect cells. Chimeric rabbit hemorrhagic disease viruses VLPs have been produced by expression of VP60 fused with hemagglutinin helper T cell epitope (HAT) at its N-terminus in insect cells and enhance the activation and proliferation of T cells [13]. Chimeric HPV-VLPs composed of L1 capsid protein fused with T-cell epitopes of HPV E6 and E7 proteins at its C-terminus have been produced in tomato plants and induce humoral and CTL responses [14].…”