RNA Vacci-Code

Protecting humans against emerging RNA viral pathogens is a crucial challenge. Vaccines –specifically live attenuated vaccines- are the most efficient way to provide rapidly lasting protection to large populations. However, producing rapidly attenuated vaccines remains difficult and quite unpredictable. We propose a renewed paradigm to conceive new-generation live attenuated RNA virus vaccines. Our strategy takes advantage of the capacity of large-scale genomic re-encoding (a method that modifies nucleic acid composition of coding regions without modifying encoded proteins) to significantly decrease viral replicative fitness. Our preliminary results show that this method can reduce the replicative fitness of alpha- & flaviviruses in cellulo and that re-encoded (RE) tick-borne encephalitis virus (TBEV) safely vaccinate susceptible mice and protect them in a challenge using wild-type (WT) pathogenic TBEV. We will start from the in silico analysis of encoding characteristics of alpha- & flaviviral genomes. Our models will be the chikungunya virus (CHIKV) & TBEV; other flaviviruses studied will include the TBEV-related hyper-virulent Omsk (OHFV) & Alkhurma (AHFV), Japanese encephalitis (JEV) and Yellow fever (YFV) viruses. Using a package of novel Java coded multi-threaded software programmes developed for the project, we will produce statistical analyses of encoding biases (nature, extent, location) to guide the design of RE genomes and experimentally decipher the mechanisms underlying the encoding process. This strategy is expected to allow weighting the roles of mechanisms previously proposed for explaining RE-based attenuation (eg modification of RNA secondary structures; dinucleotide content, codon usage, codon pair biases...), and identifying new biases, eg under- or over-representation of yet undiscovered nucleotide patterns. RE viruses will be produced and their replicative fitness and evolutionary escape strategies studied in cellulo. Selected candidates will proceed to animal experiments in which clinical & biological phenotypes will be characterised. The immunising capacity of the best RE vaccine candidates will be tested in animal experiments and compared with that provided by existing vaccines in infectious challenges using the WT viruses. The immune response will be characterised in depth in the case of TBEV by examining the induction of neutralising antibodies, characterising cell mediated immunity and analysing cytokines/chemokines molecular signatures. In the case of CHIKV & YFV, the infection of relevant Aedes vectors (Ae. aegypti; Ae. albopictus) by RE viruses will be examined. Comparative protocols will be set-up using WT & RE viruses and replication and transmission phenotypes studied for characterising vector competence. In the case of OHFV & AHFV, experimental work will be performed in a BSL4 laboratory. Il will include generation of RE viruses from molecular constructs, analysis of replicative fitness in cellulo and the use of a mouse model for studying biological & clinical phenotype and for WT virus infectious challenges. Finally, we will examine whether a RE viral backbone would be usable for transporting antigens from taxonomically related viruses. This strategy has been used previously, based on a Yellow fever 17D backbone and other flaviviral structural genes. We will compare the characteristics of genome-scale RE JEV, YFV & TBEV strains with those of chimeras in which flaviviral envelope genes will be introduced in attenuated flaviviral backbones and by inserting native or RE OHFV & AHFV structural genes in a TBEV RE backbone. Altogether, this programme will provide important experimental information relating to the strategy allowing to design rapidly RE vaccine candidates following the emergence of new, possibly hyper-virulent, RNA viral pathogens and to further understand the rules and constraint that characterise the molecular encoding of RNA viruses.