LAUSR

Novel adjuvants are highly desired to improve immune responses of SARS‐CoV‐2 vaccines. This work reports the potential of the stimulator of interferon genes (STING) agonist adjuvant, the cyclic di‐adenosine monophosphate (c‐di‐AMP), in a SARS‐CoV‐2 vaccine based on the receptor binding domain (RBD). Here, mice immunized with two doses of monomeric RBD adjuvanted with c‐di‐AMP intramuscularly were found to exhibit stronger immune responses compared to mice vaccinated with RBD adjuvanted with aluminum hydroxide (Al(OH)3) or without adjuvant. After two immunizations, consistent enhancements in the magnitude of RBD‐specific immunoglobulin G (IgG) antibody response were observed by RBD + c‐di‐AMP (mean: 15360) compared to RBD + Al(OH)3 (mean: 3280) and RBD alone (n.d.). Analysis of IgG subtypes indicated a predominantly Th1‐biased immune response (IgG2c, mean: 14480; IgG2b, mean: 1040, IgG1, mean: 470) in mice vaccinated with RBD + c‐di‐AMP compared to a Th2‐biased response in those vaccinated with RBD + Al(OH)3 (IgG2c, mean: 60; IgG2b: n.d.; IgG1, mean: 16660). In addition, the RBD + c‐di‐AMP group showed better neutralizing antibody responses as determined by pseudovirus neutralization assay and by plaque reduction neutralization assay with SARS‐CoV‐2 wild type. Moreover, the RBD + c‐di‐AMP vaccine promoted interferon‐γ secretion of spleen cell cultures after RBD stimulation. Furthermore, evaluation of IgG‐antibody titers in aged mice showed that di‐AMP was able to improve RBD‐immunogenicity at old age after 3 doses (mean: 4000). These data suggest that c‐di‐AMP improves immune responses of a SARS‐CoV‐2 vaccine based on RBD, and would be considered a promising option for future COVID‐19 vaccines.