Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

Abstract

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34°C/35°C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.negative-strand RNA virus | respiratory syncytial virus | live attenuated vaccine | codon pair deoptimization | genetic stability T he availability and affordability of large-scale custom DNA synthesis opened the new field of synthetic biology (1). The combined approach of sequence design and synthetic biology allows the generation of DNA molecules with extensive targeted modifications. Synonymous genome recoding, in which one or more ORFs of a microbial pathogen are modified at the nucleotide level without affecting amino acid coding, currently is being widely evaluated to reduce pathogen fitness and create potential live attenuated vaccines, particularly for RNA viruses (reviewed in ref.2) (3-7). The main strategies for attenuation by synonymous genome recoding are codon deoptimization (CD), codon pair deoptimization (CPD), and increasing the dinucleotide CpG and UpA content (which is usually the result of CD and CPD) (2).The mechanisms of attenuation by these strategies are currently under intensive research. It has been suggested that the primary effect of CD and CPD is to reduce translation efficiency of pathogen mRNAs, thereby providing attenuation (8). Effects on mRNA stability also can be a factor (9). In addition, recent studies suggested that codon pa...

Emergency Services of Viral RNAs: Repair and Remodeling

“…The fitness was partially restored upon serial passages, and this improvement was attributed to two factors: mutations in another viral protein, phosphoprotein (known to exhibit anti-interferon activity in the rabies virus but not in VSV), and interferon-sensitive members of the quasispecies, which were complemented by their interferon-insensitive counterparts (506). Introduction of 1,378 synonymous mutations in the ORF of the L (RdRP) gene of human respiratory syncytial virus (a pneumovirus) rendered the virus highly temperature sensitive (507). However, passages of its different lineages at stepwise increasing temperatures resulted in the selection of multiple, less-debilitated variants with mutations in various other viral proteins (in 9 of the 11 ORFs) and also in the intergenic regions.…”

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes

“…The phenotype of temperature-sensitivity plays a significant role in some live virus vaccines as, for example, in live attenuated influenza vaccines [41] or the oral poliovirus vaccine [42]. Moreover, temperature sensitivity has been previously reported as a phenotype for codon-pair-deoptimized human respiratory syncytial viruses (RSV)[26,43]. Therefore, we examined deoptimized DENV2 variants E hmin and NS3 hmin for temperature sensitivity in infected Vero cells (Fig.…”

Ongoing developments in RSV prophylaxis: a clinician’s analysis

“…These vaccines are engineered through passaging the live virus at gradually lower temperatures and deleting part of the genome. The goal is that attenuated virus maintains sufficient viral genome RNA replication to illicit enough antibody response in RSV-naïve infants, yet with a low risk of deattenuation and does not lead to the associated harmful effects [30]. Two substantial advantages of a live-attenuated vaccine are that it is, in theory, safe for RSV-naïve infants because it does not exacerbate future exposure to RSV and it can be administered intranasally.…”