Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization

Abstract

Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32°C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negativestrand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.negative strand RNA virus | pneumovirus | live attenuated vaccine H uman respiratory syncytial virus (RSV) is a negative-strand RNA virus of genus Pneumovirus, family Paramyxoviridae. RSV is the most important viral agent of serious respiratory tract illness in infants and children worldwide (1-3). Worldwide, nearly all children are infected by RSV at least once by the age of 2 y. RSV disease ranges from rhinitis to bronchiolitis or pneumonia. The RSV genome consists of a single-stranded negative-sense 15.2-kb RNA and has 10 genes in the order 3′ NS1-NS2-N-P-M-SH-F-G-M2-L 5′ (for a review, see ref. 4). The M2 gene encodes two separate proteins, M2-1 and M2-2, from overlapping ORFs.RSV vaccines and new antiviral drugs are in preclinical and clinical development; however, no RSV vaccines or antiviral drugs suitable for routine use are commercially available. The goal of the present study was to design and generate new vaccine candidates for RSV using the recently described strategy of codon-pair deoptimization (CPD) (5). By this strategy, one or more ORFs in a virus or other pathogen are recoded by rearranging existing synonymous codons so as to increase the presence of underrepresented codon pairs within the ORF. CPD can be done without changing codon use although, in the present study, codon use was occasionally changed slightly when we manually edited the sequence to remove features such as long homooligomers. Amino acid coding was completely unaffected, and nontranslated genome regions were unchanged. A major effect of CPD is...

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

“…1 i and C). By testing CPD of various mammalian viruses (16,(56)(57)(58)(59)(60) in tissue culture or in experimental animals, we have concluded that there is no single specific effect of CPD that can be related with certainty to observed deficiencies in viral proliferation of Min constructs.…”

“…The increase in CpG content (Table S4) may lead to an enhanced innate immune response in the infected cell (56,61); (ii) a robust ts phenotype (59,60); (iii) a reduction (degradation?) of the yield of viral mRNA (57) The experiments with A549 cells were revealing because an inhibitor (BX795) of the innate immune response (30,31) reversed to some extent the low si values in these cells of the three viruses tested.…”

Synonymous Deoptimization of Foot-and-Mouth Disease Virus Causes Attenuation In Vivo while Inducing a Strong Neutralizing Antibody Response

“…Since deoptimization is due to the summation effect at each of hundreds or thousands of nucleotide mutations without changing amino acid sequences, the likelihood of reversion to virulence is expected to be reduced. Indeed, this technology has proven useful to derive attenuated strains of positive-strand RNA viruses such as poliovirus (16)(17)(18), porcine reproductive and respiratory syndrome virus (PRRSV) (29), and dengue virus (26), as well as those of negative-sense RNA viruses such as influenza virus (20), respiratory syncytial virus (24), and vesicular stomatitis virus (27).…”

“…However, alternative mechanisms involving the induction of innate immunity as a result of changing dinucleotide frequency have been proposed by other authors (23). Furthermore, codon pair bias deoptimization of other viruses, including respiratory syncytial virus (RSV) (24), human immunodeficiency virus type 1 (25), dengue virus (26), and vesicular stomatitis virus (27), have also led to attenuated strains. All these reports indicate that codon and codon pair bias deoptimization can also be a useful tool to develop attenuated vaccine candidates against RNA viruses.…”

A Recombinant Respiratory Syncytial Virus Vaccine Candidate Attenuated by a Low-Fusion F Protein Is Immunogenic and Protective against Challenge in Cotton Rats

“…One such genetic modification we recently described is the codon deoptimization of RSV nonstructural proteins NS1 and NS2 (dNS), which are virulence proteins that antagonize the host interferon responses (7). Codon deoptimization (8,9) and codon pair deoptimization (10,11) are strategies to decrease viral protein expression by incorporating the least used codons or least used codon pairs in the human genome, respectively. In previous studies, deletion of NS1 was overattenuating in nonhuman primates (12), whereas deletion of NS2 was underattenuating (13).…”