ABSTRACT
An inability to proliferate at high temperatures typically gives viruses an attenuated phenotype. Here, we present a protocol to obtain and isolate temperature-sensitive (TS) SARS-CoV-2 strains via 5-fluorouracile-induced mutagenesis. We describe steps for the induction of mutations in the wild-type virus and selection of TS clones. We then show how to identify the mutations associated with the TS phenotype, following forward and reverse genetics strategies. For complete details on the use and execution of this protocol, please refer to Yoshida et al. (2022).1.
ABSTRACT
Live-attenuated vaccines are generally highly effective. Here, we aimed to develop one against SARS-CoV-2, based on the identification of three types of temperature-sensitive (TS) strains with mutations in nonstructural proteins (nsp), impaired proliferation at 37°C-39°C, and the capacity to induce protective immunity in Syrian hamsters. To develop a live-attenuated vaccine, we generated a virus that combined all these TS-associated mutations (rTS-all), which showed a robust TS phenotype in vitro and high attenuation in vivo. The vaccine induced an effective cross-reactive immune response and protected hamsters against homologous or heterologous viral challenges. Importantly, rTS-all rarely reverted to the wild-type phenotype. By combining these mutations with an Omicron spike protein to construct a recombinant virus, protection against the Omicron strain was obtained. We show that immediate and effective live-attenuated vaccine candidates against SARS-CoV-2 variants may be developed using rTS-all as a backbone to incorporate the spike protein of the variants.