Attenuation and Stability of CHIKV-NoLS, a Live-Attenuated Chikungunya Virus Vaccine Candidate

Our previous investigation of the nucleolar localisation sequence (NoLS) of chikungunya virus (CHIKV) capsid protein demonstrated the role of capsid in CHIKV virulence. Mutating the NoLS of capsid in CHIKV led to the development of a unique live-attenuated CHIKV vaccine candidate, termed CHIKV-NoLS. CHIKV-NoLS-immunised mice developed long-term immunity from CHIKV infection after a single dose. To further evaluate CHIKV-NoLS attenuation and suitability as a vaccine, we examined the footpad of inoculated mice for underlying CHIKV-NoLS-induced immunopathology by histological and flow cytometric analysis. In comparison to CHIKV-WT-infected mice, CHIKV-NoLS-inoculated mice exhibited minimal inflammation and tissue damage. To examine the stability of attenuation, the plaque phenotype and replication kinetics of CHIKV-NoLS were determined following extended in vitro passage. The average plaque size of CHIKV-NoLS remained notably smaller than CHIKV-WT after extended passage and attenuated replication was maintained. To examine thermostability, CHIKV-NoLS was stored at 21 degrees C, 4 degrees C, -20 degrees C and -80 degrees C and infectious CHIKV-NoLS quantified up to 84 days. The infectious titre of CHIKV-NoLS remains stable after 56 days when stored at either -20 degrees C or -80 degrees C. Interestingly, unlike CHIKV-WT, the infectious titre of CHIKV-NoLS is not sensitive to freeze thaw cycles. These data further demonstrate preclinical safety and stability of CHIKV-NoLS.