Immunogenicity and safety of a live-attenuated SARS-CoV-2 vaccine candidate based on multiple attenuation mechanisms

mRNA vaccines against SARS-CoV-2 were rapidly developed and were effective during the pandemic. However, some limitations remain to be resolved, such as the short-lived induced immune response and certain adverse effects. Therefore, there is an urgent need to develop new vaccines that address these issues. While live-attenuated vaccines are a highly effective modality, they pose a risk of adverse effects, including virulence reversion. In the current study, we constructed a live-attenuated vaccine candidate, BK2102, combining naturally occurring virulence-attenuating mutations in the NSP14, NSP1, spike, and ORF7-8 coding regions. Intranasal inoculation with BK2102 induced humoral and cellular immune responses in Syrian hamsters without apparent tissue damage in the lungs, leading to protection against a SARS-CoV-2 D614G and an Omicron BA.5 strains. The neutralizing antibodies induced by BK2102 persisted for up to 364 days, which indicated that they confer long-term protection against infection. Furthermore, we confirmed the safety of BK2102 using transgenic (Tg) mice expressing human ACE2 (hACE2) that are highly susceptible to SARS-CoV-2. BK2102 did not kill the Tg mice, even when virus was administered at a dose of 10⁶ plaque-forming units (PFUs), while 10² PFU of the D614G strain or an attenuated strain lacking the furin cleavage site of the spike was sufficient to kill mice. These results suggest that BK2102 is a promising live-vaccine candidate strain that confers long-term protection without significant virulence.