Construction Of A Live-Attenuated CVB3 Vaccine Through Amber Suppression

Coxsackievirus B3, a member of the genus Enterovirus within the family Picornaviridae, is a primary causative agent of virus-induced myocarditis and dilated cardiomyopathy, especially in young infants. Between 5-50% of myocarditis and its end-stage, dilated cardiomyopathy (DCM), are attributable to CVB3 infection. Unfortunately, there is no specific therapeutic agent available to address expansion CVB3-induced myocarditis and no vaccine for clinical use.Amber suppression means translation system reads through the amber codon (UAG) in the mRNA, which would be used to control the synthesis of intact protein and expand genetic code by incorporatin of unnatural amino acids. The fidelity of natural protein synthesis is maintained by specific aminoacylation of a transfer RNA (tRNA) with an amino acid, and the ribosomal decoding of each tRNA in response to a cognate codon on a messenger RNA (mRNA). Amber suppression uses an ’orthogonal’ aminoacyl tRNA synthetase-tRNA pair (that is, a synthetase that does not aminoacylate the normal tRNA range of a cell and a tRNA that is not a substrate for the normal synthetase range of the cell) to direct the incorporation of a specific amino acid in response to an amber stop codon in a gene of interest. When a triplet codons in a gene of interest has been instead by an amber nonsense codon, the synthesis of interest intact protein (such as the protein of virus) will be controlled by the in present or absent of amber suppression system. The application of amber suppression in virology facilitates the study of new type vaccine.Here we report a new way to construct a live-attenuated CVB3 vaccine through amber suppression. We envisaged that if we could introduce blank codons at positions within the CVB3 genome that encode essential proteins, we would prevent proper translation and assembly replicate, thus making a safe vaccine. The amber suppression system would also allow us to turn on and off multiple replication cycles of CVB3 in future vaccine development in humanized mouse.To construct the amber suppression system, special TyrRS-tRNA derived fromE.coli, which is orthogonal function in eukaryon expression system, are firstly cloned into plasmid pcDNA3.1(+). In this amber suppression expression vector, TyrRS was cloned into multiple cloning site and H1 promoter driven tRNACUATyr expression cassette (H1-tRNACUATyr) was inserted at CMV promoter upstream reversely and repeated four times in tandem to increase tRNA expression levels. And then we identified and assayed the efficiency of EcTyrTS/tRNA amber suppression system by expression of enhanced green fluorescent protein containing a Tyr39UAG amber mutant, which finally expression levels is 28.4% of wild type EGFP.Subsequently, we site-specific mutate the Tyr27 codon on the VP4 of CVB3 protein-encoding gene into an amber codon. The in vitro transcript mRNA of mutated CVB3 was co-transfected into 293T cells with plasmid pcDNA3.1-EcYRS/tRNA. After three times virus blind passage, the virus-induced cytopathic effect was faintly observed only when the RS/tRNA pair in presence.Taken together, our work, for the first time, explored the application of amber suppression for constructing a safe vaccine of picornaviruse, specifically CVB3. We have demonstrated the precise control of CVB3 viability in vitro can be achieved by using an amber suppression strategy. The above work is an important progress towards the development of a safe and effective CVB3 vaccine. Also this work inspired the study of virus shell-engineering as the re-program of unnatural amino acids contain unique functional groups into amber codon allow selective modification of viral proteins through bio-orthogonal reactions, which facilitate evaluation of vaccine in turn. For higher production level of intact recombined CVB3-UAG virion, we are currently working on constructing a lentiviral-based gene delivery system to stably suppress amber codon in susceptible cells, specifically HeLa cells. We envisaged that the susceptible cells will tolerate the amber suppression and then produce CVB3-UAG virion in higher lever.