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

Abstract

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 °C, 4 °C, -20 °C and -80 °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 °C or -80 °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.

Keywords: alphavirus; chikungunya virus; live-attenuated; preclinical; vaccine.

Figure 1

Histological analysis of the mouse footpad after inoculation with 10⁴ plaque forming units (PFU) CHIKV-WT or CHIKV-NoLS. Twenty-eight-day-old C57BL/6 mice were inoculated in the ventral/lateral side of the right foot with 10⁴ PFU CHIKV-WT or CHIKV-NoLS or mock-infected with (phosphate buffered saline) PBS alone. (a) CHIKV-induced footpad swelling was assessed daily by measuring the height and width of the perimetatarsal area of the hind foot. Each symbol represents the mean ± standard error from 5 mice; (b) Inoculated (ipsilateral) feet were dissected, processed for histological analysis and stained with H&E. Images at 100× and 200× magnification are representative of at least 6 fields of view. Black arrows indicate cellular infiltration. The black dashed box indicates the area magnified at 200×. The size bars represent 100 µm; (c) Cellular infiltrates at 200× magnification were quantified using ImageJ software. ***, p < 0.001, ns―not significant. Statistical analyses were performed using one-way ANOVA with Tukey’s post-test. Data represents the mean ± standard error.

Figure 2

Cellular infiltrates in the feet of inoculated mice were analysed using flow cytometry. (a) Total live CD45⁺ leukocytes, (b) CD4⁺ and CD8⁺ T cells (CD3⁺), (c) inflammatory monocytes (Ly6C^hi Ly6G^- CD11b^hi MHC-II⁺), (d) Neutrophils (Ly6G^hi CD11b^hi) and (e) tissue macrophages (Ly6C^lo CD11b^hi Ly6G^- MHC-II⁺). *, p < 0.05, **, p < 0.01, and ****, p < 0.0001. Statistical analyses were performed using a Mann–Whitney U-test (A, B, C, D) or a a two-way ANOVA with a Holm-Sidak comparison correction. Data represents the mean ± standard error.

Figure 3

Multistep growth kinetics of CHIKV-NoLS in C6/36 cells after extended in vitro passage. C6/36 cells were infected with CHIKV-WT, P0 CHIKV-NoLS or P5 CHIKV-NoLS at an MOI of 0.1 PFU/cell. Supernatants were collected at the indicated time points, and infectious virus was quantified by plaque assay. **, p < 0.01, and ***, p < 0.001. Statistical analyses were performed using two-way ANOVA with Bonferroni post-tests. Each symbol represents the mean ± standard error from 3 independent experiments.

Figure 4

CHIKV-NoLS plaque size after extended in vitro passage. CHIKV-NoLS and CHIKV-WT were passaged 10 times in Vero cells at 37 °C using a multiplicity of infection of 0.1 PFU/cell. With each passage of virus, Vero cells were infected with 30 PFU and plaque size measured using ImageJ software. Each dot represents one plaque. Dashed lines represent the mean plaque size for CHIKV-NoLS and CHIKV-WT at passage 0.

Figure 5

Long term thermostability of CHIKV-NoLS. CHIKV-NoLS and CHIKV-WT were diluted to 5 × 10⁵ pfu/ml and vials stored at 21 °C, 4 °C, −20 °C and −80 °C. At the indicated time points infectious CHIKV-NoLS and CHIKV-WT was quantified by plaque assay. Each symbol represents the mean ± standard error.

Figure 6

Stability of CHIKV-NoLS infectivity following repeated freeze thaw cycles. CHIKV-NoLS and CHIKV-WT were diluted to 5 × 10⁵ PFU/mL and vials stored at −80 °C. Vials were thawed on ice and infectious CHIKV-NoLS and CHIKV-WT quantified by plaque assay. Vials were returned to −80 °C and the process repeated. Each symbol represents the mean ± standard error.