Chikungunya Virus Strains Show Lineage-Specific Variations in Virulence and Cross-Protective Ability in Murine and Nonhuman Primate Models

Abstract

Chikungunya virus (CHIKV) is a reemerging arbovirus capable of causing explosive outbreaks of febrile illness, polyarthritis, and polyarthralgia, inflicting severe morbidity on affected populations. CHIKV can be genetically classified into 3 major lineages: West African (WA); East, Central, and South African (ECSA); Indian Ocean (IOL); and Asian. Additionally, the Indian Ocean (IOL) sublineage emerged within the ECSA clade and the Asian/American sublineage emerged within the Asian clade. While differences in epidemiological and pathological characteristics among outbreaks involving different CHIKV lineages and sublineages have been suggested, few targeted investigations comparing lineage virulence levels have been reported. We compared the virulence levels of CHIKV isolates representing all major lineages and sublineages in the type I interferon receptor-knockout A129 mouse model and found lineage-specific differences in virulence. We also evaluated the cross-protective efficacy of the IOL-derived, live-attenuated vaccine strain CHIKV/IRESv1 against the Asian/American CHIKV isolate YO123223 in both murine and nonhuman primate models, as well as the WA strain SH2830 in a murine model. The CHIKV/IRES vaccine provided protection both in mice and in nonhuman primate cohorts against Caribbean strain challenge and protected mice against WA challenge. Taken together, our data suggest that Asian/American CHIKV strains are less virulent than those in the Asian, ECSA, and WA lineages and that despite differences in virulence, IOL-based vaccine strains offer robust cross-protection against strains from other lineages. Further research is needed to elucidate the genetic basis for variation in CHIKV virulence in the A129 mouse model and to corroborate this variation with human pathogenicity.IMPORTANCE Chikungunya virus (CHIKV) is a reemerging human pathogen capable of causing debilitating and disfiguring polyarthritis, which can last for months to years after initial fever has resolved. There are four major genetic lineages of CHIKV, as well as two recently emerged sublineages, none of which have been evaluated for differences in virulence. Moreover, the ability of chikungunya vaccines to cross-protect against heterologous CHIKV lineages has not been explored. Therefore, we sought to compare the virulence levels among CHIKV lineages, as well as to evaluate the cross-protective efficacy of the CHIKV/IRESv1 vaccine candidate, in two different models of CHIKV infection. Our results suggest that, although significant differences in virulence were observed among CHIKV lineages, the CHIKV/IRESv1 vaccine elicits cross-lineage protective immunity. These findings provide valuable information for predicting the severity of CHIKV-associated morbidity in future outbreaks, as well as vaccine development considerations.

Keywords: alphavirus; chikungunya; vaccine.

FIG 1

Survival of CHIKV-infected A129 mice differs by lineage. Four- to 8-week-old A129 mice were infected with approximately 10⁴ PFU of a CHIKV isolate from one of 5 genetic CHIKV lineages (n = 5 or 6 per group), and survival was assessed, with euthanasia counting as a death on the following day. Isolates were pooled into their respective groups, and survival curves were analyzed by Kaplan-Meier survival analysis using log rank test in SigmaPlot with the Holm-Sidak method of multiple pairwise comparisons. WA strains induced a significant leftward shift in the survival curve compared with all other lineages, while Asian/American strain-infected mice survived significantly longer than mice infected with strains from WA, ECSA, and Asian lineages (P << 0.005, denoted by **); survival was not significantly different between ECSA-, IOL-, and Asian lineage-infected mice.

FIG 2

Comparison of viremia, weight, and footpad height changes between A129 mice infected with CHIKV isolates from different lineages. Four- to 8-week-old A129 mice were infected with approximately 10⁴ PFU of a CHIKV isolate from one of 5 genetic CHIKV lineages (n = 5 or 6 per strain/isolate), blood was taken on days 1 to 3 from alternating mice to assess viremia (A), and weight (B) and footpad height (C) were monitored daily; data were pooled by lineage for analysis. A129 mice infected with WA CHIKV isolates had significantly higher viremia than those infected with ECSA and IOL isolates 1 day postinfection and those infected with ECSA, IOL, and Asian/American isolates 2 days postinfection (ANOVA, P < 0.05 denoted by *). Weight change was not significantly different (one-way ANOVA, P = 0.32), while footpad height change was significantly different 2 to 5 DPI (one-way ANOVA, P >> 0.05, denoted by **), with IOL strains inducing significantly more footpad swelling on day 2 (one-way ANOVA with Bonferroni post hoc test, P < 0.01).

FIG 3

CHIKV vaccine candidate CHIKV/IRESv1 at multiple doses provides complete protection from lethal YO123223 and SH2830 CHIKV challenge in adult A129 mice. (A and B) A129 mice were vaccinated with various doses of CHIKV/IRESv1 vaccine or PBS and then challenged 4 weeks later with Asian/American CHIKV strain YO123223, and weight (A) and footpad height (B) were monitored until all mice in the sham-vaccinated group succumbed to infection. (C and D) A129 mice were vaccinated with either 10³ or 10⁵ PFU of CHIKV/IRESv1 vaccine or PBS and then challenged with West African CHIKV strain SH2830, and weight (C) and footpad height (D) were monitored for 5 days.

FIG 4

Average fever hours in CHIKV/IRESv1-vaccinated and naive cynomolgus macaques after CHIKV YO123223 challenge. Fever hours are defined as the number of hours within a 24-h time period in which core temperature exceeded 1.5 times the maximum standard deviation of the control temperature value determined prior to challenge. (A) Sham group vaccinated with saline; vaccine dose (in PFU) received by group denoted in color key. (B) Inset graph shows representative sample of core temperature changes (Δ) in sham-vaccinated animals subcutaneously challenged with CHIKV YO123223 or LR2006 OPY1 virus, both at 1.0E+5 PFU. Data were time matched to preexposure values from each animal for derivation of relative temperature change.

FIG 5

IOL-based CHIKV/IRESv1 CHIKV vaccine prevents viremia in vaccinated cynomolgus macaques challenged with CRBN isolate YO123223. Viremia of cynomolgus macaques vaccinated with CHIKV/IRESv1 prior to viral challenge with CHIKV YO123223 for animals sham vaccinated with saline (red circles; light red triangles indicate historical data for challenge with LR2006) or a prime-only intradermal (100-µl) dose of CHIKV/IRESv1 at either 5.0E+3 (light blue circles), 1.0E+4 (medium blue circles), or 5.0E+4 (dark blue circles) PFU. Dashed line indicates limit of detection (LOD) of plaque assay.