Seminars

We present a comprehensive investigation into SARS-CoV-2 transcription regulatory sequences (TRS) and their application in developing a novel live attenuated vaccine that provides variant-independent protection with robust mucosal immunity.

Through systematic evaluation of TRS elements, we identified ORF7a as possessing the strongest transcriptional activity. Our findings reveal that the core TRS sequence (ACGAAC) requires both 5' and 3' flanking sequences for optimal gene expression, with secondary structure integrity being critical for function.

Leveraging these insights, we developed N-fused reporter viruses (Nf) and incorporated targeted nsp1 mutations to create the NfCtQF strain. This vaccine candidate achieved complete attenuation in hACE2-transgenic mice while maintaining strong immunogenicity. Critically, a single dose of NfCtQF provided complete protection against lethal challenge with both the original SARS-CoV-2 strain and Omicron variants (BA.1 and XBB.1.5) without requiring variant-specific vaccine updates.

The vaccine's key advantage lies in its ability to elicit robust neutralizing antibodies in both systemic and mucosal compartments, effectively blocking viral replication at primary infection sites. Unlike current vaccines that primarily induce systemic immunity, our live attenuated approach ensures mucosal protection at the nasal turbinates and lungs—the critical first line of defense against respiratory viruses.

This genetically stable, BSL-2 platform offers a transformative approach to pandemic preparedness, providing durable cross-variant protection through a single immunization while establishing the mucosal immunity essential for preventing viral transmission.