Hybrid Methods Guide Structure Based Vaccine Design for Picornaviruses

Abstract

The physical properties of viral capsids are major determinants of vaccine efficacy for picornaviruses which impact on human and animal health. Current vaccines are produced from inactivated virus. Inactivation often reduces the stability of the virus capsid, causing a problem for Foot and Mouth Disease Virus (FMDV) where certain serotypes fall apart into pentameric assemblies below pH 6.5 or at temperatures above 37°C, destroying their effectiveness in eliciting a protective immune response. As a result, vaccines require a cold chain for storage and animals need to be frequently immunised. Globally there are seven FMDV serotypes: O, A, Asia1, C and SAT-1, -2 and -3, contributing to a dynamic pool of antigenic variation. We sought to rationally engineer FMDV capsids either as infectious copy virus or recombinant empty capsids with improved thermo-stability for improved vaccines. Here we used in-silico MD simulations, molecular modelling, free energy calculations, X-ray crystallography, Cryo-electron microscopy (CryoEM) and various biochemical/biophysical techniques to design and help characterise the improved capsids. For the most unstable FMDV serotypes (O and SAT2), panels of stabilising mutants were characterised. Promising candidates were then engineered and shown to confer increased thermo- and pH-stability. Thus, in-silico predictions translate into marked stabilisation of both infectious and recombinant empty capsids.

An in-situ diffraction method was used to determine crystal structures to verify that no unanticipated structural changes have occurred as a consequence of the modifications made. Where it was difficult to obtain crystals/diffraction, structures were determined by high-resolution CryoEM (with the best electron density maps reaching 2.7Å resolution). The structures of the wildtype and two of the stabilised mutants for three different serotypes of FMDV showed the mutations made predicted interactions and the antigenic surfaces remained unchanged.

Animal trials showed stabilised particles can generate improved neutralising response compared to the traditional vaccines. Similar approach applied to the polio virus successfully produced antigenic VLPs using the plant based expression system. CryoEM reconstruction of polio VLPs produced 3.6Å resolution maps and the structure analysis suggested the plant based polio particles are identical to the native virus. We have successfully used a structure based rational engineering approach to increase the stability of viral capsids without affecting the antigenicity and demonstrated the direct application of structural biology and structure based design that has the potential to lead directly to a new generation of efficacious vaccines that can provide hope that the disease can be brought under control.

In addition, using CryoEM, CryoET and Focus Ion Beam milling of the infected cells, we are working towards understanding the picronavirus life cycle in molecular details. To this end, using localised reconstruction, we have determined the interaction between αvβ6 and two FMDV strains at high resolution. In the preferred mode of engagement the fully open form of the integrin, hitherto unseen at high-resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role. Finally using Ion Beam milling, we have begun to visualise and understand the formation of virus production factories inside the infected cells. In-situ high resolution structures will allow us to see the virus life cycle in its native state and also lead to potential new targets for next generation vaccine design.

Biography

Abhay Kotecha is a senior research associate at the Division of Structural Biology, Nuffield Department of Clinical Medicine, University of Oxford, UK. Dr. Kotecha obtained his Bachelor’s degree with Honours in Cell and Molecular Biology from Oxford Brookes University, Oxford in June 2008 and his DPhil in Clinical medicine from University of Oxford in March 2013. During his studies, Dr. Kotecha worked in several world class laboratories and received many fellowships, including EMBO summer research studentship to learn advanced electron microscopy techniques at the Electron Microscopy Core Facility, EMBL, Heidelberg, Germany. BBSRC/STFC research fellowship to spend a year at Science and Technology Facilities Council, Synchrotron light source, Daresbury, UK where he worked on membrane protein X-ray crystallography of light harvesting complexes. Undergraduate research studentship, Imperial College, London. Dr. Kotecha was awarded the prestigious Wellcome Trust DPhil studentship in structural biology at the University of Oxford and the Clarendon Fund Scholarship from Oxford University Press to study viruses in atomic details. His research interests are on structure based vaccine design for infectious viruses using X-ray crystallography and Cryo-Electron Microscopy. His work funded by The Wellcome Trust Translational Award and Gates Foundation aimed at developing stable synthetic vaccines. He has successfully developed novel empty particle vaccines for FMDV which is now being taken up by commercial partners for industrial production. This work has a very broad national and international impact and may also be applicable across a range of human and animal pathogens. Dr. Kotecha has published more than 20 papers in high impact journals most of which has received national and international media coverage.

He is now investigating the stabilised virus particles in cellular context to understand the virus receptor interaction, disassembly of stabilised viruses and the genome release as well as the assembly of newly synthesised particles to generate new targets for next generation of vaccines.