Supporting data for "Generation of Live Attenuated Vaccines against SARS-CoV-2"

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged during late 2019 to take the whole world by surprise, prompting the declaration of pandemic, COVID-19. As mentioned in the name, it is known to cause respiratory diseases and has infected majority of the human population worldwide. Early therapeutic options of SARS-CoV-2 focused on the remedial purposes to alleviate severe disease, and later, prophylactic vaccines against SARS-CoV-2 were developed. There are several vaccine categories, the well-known SARS-CoV-2 vaccines include nucleic acid, viral vectored and inactivated vaccines. Since then, SARS-CoV-2 has taken a new turn to evolve into Omicron variant to evade vaccine-induced immune responses, rendering existing vaccines ineffective in preventing illness. Live attenuated vaccines were previously proven to eradicate or nearly eradicate pathogenic diseases, including smallpox and polio, respectively. Its capability to induce mucosal and sterilising immunity prevents viral pathogenesis and shedding, contributing to herd immunity.

Coronaviruses contain conserved motifs that spans throughout the family, which are commonly used for vaccine development. First, we targeted the 2’-O-methyltranferase protein by catalytically inactivation through a single point mutation, designated as d16. Then, we displayed its safety profile and immunogenicity. Thereon after, we shown that a single dose of d16 could effectively protect the host against SARS-CoV-2 ancestral strain challenge through in vivo models, Syrian hamster and K18-hACE2 transgenic mice. However, we noticed minor inflammatory response among vaccinated hamster lung tissues and single point mutation faces risk of reversion mutation. Our previous studies showed that SARS-CoV ORF3a is involved in NLRP3 inflammasome activation by promoting TRAF-3 dependent ubiquitination of p105 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). We sought to downregulate ORF3a-associated inflammation by inversion abrogation of ORF3a genome in the bacterial artificial chromosome (BAC), designated d16i3a. We have shown the impeded pathogenicity while maintaining excellent vaccine efficacy of d16i3a against various SARS-CoV-2 variants. Additionally, d16i3a vaccination resulted in robust T cell activation and could potentially serve as a booster vaccine to complement existing vaccination regimens. Omicron variants continuously adapt and mutate to avert host immune responses, we strived to construct an Omicron-specific d16i3a live attenuated vaccine. Hence, the ancestral spike of d16i3a is replaced with spike sequence of Omicron XBB.1.16, designated d16i3a-XBB.1.16. We have shown that d16i3a-XBB.1.16 could retrospectively protect against BA.5 variant as well as prospective SARS-CoV-2 variant, JN.1 in animal models. This study highlights the development of a potential SARS-CoV-2 live attenuated vaccine to put an end to the endemic.