Seminar

Rational engineering of Picornavirus capsids to produce stable vaccines

Abhay Kotecha, PhD

The physical properties of viral capsids are major determinants of vaccine efficacy for several picornaviruses important for human and animal health. Current picornavirus vaccines consist of intact virus capsids produced usually from inactivated virus. The inactivation may have a detrimental effect on the stability of capsids. This is an especial problem for the Foot and Mouth disease virus (FMDV) where capsids are very sensitive to pH and temperature. The viruses fall apart into pentameric assemblies below pH 6.5, presumably to facilitate uncoating after entering the host cell. Similarly, they often dissociate into pentamers at little above 37°C. This has a major consequence since one of the most important factors in the potency of inactivated virus vaccines is the integrity of virus capsids. As a result vaccines requires a cold chain and frequent immunisation of animals is required.In this project we combined in silico molecular dynamics simulation with experimental crystallographic validation and novel methods for measuring thermostabilityto rationally engineer thermo-stable FMDV capsids. We have demonstrated that in silico predictions translate into marked stabilisation of both infectious and empty viral capsids. Crystallography was used for quality assessment and to verify that the intended mutations are present and that no unanticipated structural changes have occurred as a consequence of the modifications made that might impact on the immunogenicity of the particles. We used a relatively high-throughput In-situ diffraction method to collect high-resolution datafrom micro-crystals directly from the crystallisation drops within the crystallisation plates (1). The structures of the wild type and the stabilised mutants were solved and found to be identical.The In-situ technology is a quick and easy method for collecting room temperature data, avoiding the increased mosaicityoften associated withcryo-crystallography. It also provides a contained environment faciliating safe data collection and avoiding the hazards and deleterious effects of crystal mounting. A sufficiently complete data set can often be collected form a single 96-well, diffraction-optimised plate. Stabilised capsids have been used in animal trials and found to generate increased neutralising antibody response. Such capsids will likely form the basis of the next generation of safe vaccines against the disease. (1) Axford D, et al,ActaCrystallogr D BiolCrystallogr. 2012,68:592-600.