Скачать или смотреть M. Ryan - Rational design of attenuated FMDV vaccines by elevation of –CPG- and –UPA-Dinucleotide f.

M. Ryan - Rational design of attenuated FMDV vaccines by elevation of –CPG- and –UPA- Dinucleotide frequencies

Session: The future of FMD vaccines

Open Session of the EuFMD – 2018 – Increasing Global Security in the supply of effective vaccines – 29-31 October 2018 -Borgo Egnazia, Italy

Introduction
Live, attenuated, vaccines have classically been developed by serial passage of virus in tissue-cultured cells: progeny viruses are analysed for attenuation throughout this process. Often, attenuation is based upon a small number of key mutations which may back-mutate during vaccine production, or, following administration: reversion to virulence. Modern molecular biology, in combination with reverse genetics, has facilitated FMDV attenuation by deletion/mutation of virus proteins, or, by transposition of RNA secondary structures. An alternative method - Synthetic Attenuated Virus Engineering (SAVE) - produces attenuated viruses by the rational design of genomes to include high numbers of synonymous mutations. Initially attributed to alteration of codon-pair bias, it has been shown that attenuation is due to elevation of –CpG-/–UpA- dinucleotide frequencies.

Materials and methods
Using synthetic biology we have produced FMDV ‘replicon’ systems which allow us to quantify RNA replication in real-time, in live cells. Our replicons allow the rapid determination of replicative fitness and, constructed as ‘cassette’ systems, allows genomic regions to easily be replaced. In this manner the various strategies of FMDV attenuation have been compared.

Results
Data will be presented comparing the replicative fitness of a wide range of published modified FMDV genomes, together with our SAVE genomes, in a range of cell-types. Replicons have been converted into infectious copies and virus rescued.

Discussion
Attempts in the 1960s to produce live, attenuated, FMDV vaccines via the ‘classical’ method were unsuccessful – most probably through a lack of genetic stability. Attenuation by SAVE, however, involves high numbers (many 100s) of such mutations providing a very much higher degree of stability. Our attenuation strategy is the introduction of synonymous mutations into the replication proteins (alone) producing a ‘pre-attenuated’ genomic ‘backbone’ into which capsid proteins from any sub-type can be inserted to produce a vaccine: a rapis response to new outbreak strains.

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