Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the Δorf2-8, Δorf2.4, and Δorf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the Δorf2-8 and Δorf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the Δorf2.4-puro replicon by inserting a puromycin-resistant gene into the Δorf2.4 replicon. We observed the more sustained replication of the Δorf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities.

Construction and evaluation of a self-replicative RNA vaccine against SARS-CoV-2 using yellow fever virus replicon.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global threat. To forestall the pandemic, developing safe and effective vaccines is necessary. Because of the rapid production and little effect on the host genome, mRNA vaccines are attractive, but they have a relatively low immune response after a single dose. Replicon RNA (repRNA) is a promising vaccine platform for safety and efficacy. RepRNA vaccine encodes not only antigen genes but also the genes necessary for RNA replication. Thus, repRNA is self-replicative and can play the role of an adjuvant by itself, which elicits robust immunity. This study constructed and evaluated a repRNA vaccine in which the gene encoding the spike (S) protein of SARS-CoV-2 was inserted into a replicon of yellow fever virus 17D strain. Upon electroporation of this repRNA into baby hamster kidney cells, the S protein and yellow fever virus protein were co-expressed. Additionally, the self-replication ability of repRNA vaccine was confirmed using qRT-PCR, demonstrating its potency as a vaccine. Immunization of C57BL/6 mice with 1 µg of the repRNA vaccine induced specific T-cell responses but not antibody responses. Notably, the T-cell response induced by the repRNA vaccine was significantly higher than that induced by the nonreplicative RNA vaccine in our experimental model. In the future, it is of the essence to optimize vaccine administration methods and improve S protein expression, like protection of repRNA by nanoparticles and evasion of innate immunity of the host to enhance the immune-inducing ability of the repRNA vaccine.