Chimeric alphavirus vaccine candidates for chikungunya

Abstract

Chikungunya virus (CHIKV) is an emerging alphavirus that has caused major epidemics in India and islands off the east coast of Africa since 2005. Importations into Europe and the Americas, including one that led to epidemic transmission in Italy during 2007, underscore the risk of endemic establishment elsewhere. Because there is no licensed human vaccine, and an attenuated Investigational New Drug product developed by the U.S. Army causes mild arthritis in some vaccinees, we developed chimeric alphavirus vaccine candidates using either Venezuelan equine encephalitis attenuated vaccine strain TC-83, a naturally attenuated strain of eastern equine encephalitis virus (EEEV), or Sindbis virus as a backbone and the structural protein genes of CHIKV. All vaccine candidates replicated efficiently in cell cultures, and were highly attenuated in mice. All of the chimeras also produced robust neutralizing antibody responses, although the TC-83 and EEEV backbones appeared to offer greater immunogenicity. Vaccinated mice were fully protected against disease and viremia after CHIKV challenge.

Fig. 1

Diagram of the genetic composition of the chimeric CHIKV vaccine candidates and the specific infectivities of transcribed RNAs following electroporation of BHK cells. nsP1–4, nonstructural proteins 1–4; SG, subgenomic promoter; C, capsid; E1 and E2, envelope glycoproteins 1 and 2.

Fig. 2

Expression of structural proteins by chimeric vaccine candidates. BHK-21 cells were infected with SINV Toto1101, VEEV TC-83, VEE/CHIKV, EEE/CHIKV and SIN/CHIKVD9 at an MOI of 20 PFU/cell. At 16 h post infection, cells were washed three times with PBS and incubated for 1 h at 37°C in DMEM lacking methionine and supplemented with 0.1% FBS and 20 mCi/ml of [³⁵S]methionine. After this incubation, cells were scraped into PBS, pelleted by centrifugation and dissolved in standard gel-loading buffer. Equal amounts of proteins were loaded onto sodium dodecyl sulfate (SDS)–10% polyacrylamide gels. Markers indicate p62, E1+E2 (envelope glycoproteins E1 and E2), and capsid.

Fig. 3

Histopathology and immunohistochemistry of 5-week-old C57/BL6 mouse brains following IN infection with the Ross strain of CHIKV. H&E staining of mouse brains at day 7 after IN infection with either PBS (A) or CHIKV (B). CHIKV-infected mice showed severe, multifocal inflammation and liquefactive necrosis in the cerebral cortex. Perivascular cuffs were also located diffusely throughout the cerebral cortex (arrow). Immunohistochemical staining of the cerebral cortex at day 7 after IN infection with CHIKV (C, D). Primary antibodies against Modoc virus (MODV) were used as a negative control (C). Using primary antibodies against CHIKV, there was intense positive staining (dark red) of degenerating neurons located in the necrotic areas of the cerebral cortex (D).

Fig. 4

Body weights in 3-week-old female C57BL/6 mice after SC vaccination with EEE/CHIKV, TC-83/CHIKV or SIN/CHIKV at doses of 5.8, 5.6 and 5.8 log₁₀ PFU/mouse, respectively, or of 3-week-old female NIH Swiss mice after SC vaccination with EEE/CHIKV, TC-83/CHIKV or SIN/CHIKV at doses of 5.8, 5.6 and 5.8 log₁₀ PFU/mouse, respectively.

Fig. 5

Mortality in 6-day-old NIH Swiss mice after IC infection with ca. 10⁶ PFU of chimeric CHIKV vaccine candidates, vaccine strain 181/25, or the wt CHIKV LR strain. Other alphavirus vaccine strains including VEEV strains TC-83, V3526, and chimeric SIN/VEEV and SIN/EEEV are included for comparison.

Fig. 6

Replication of CHIKV chimeric vaccine candidates or the wt LR CHIKV strain after SC infection of 3–4-day-old NIH Swiss mice with ca. 10⁵ PFU. The limits of detection are indicated by the dashed lines.

Fig. 7

Body weight of vaccinated and sham-vaccinated mice after CHIKV challenge. Cohorts of five 3-week-old females were vaccinated with EEE/CHIKV, TC-83/CHIKV or SIN/CHIKV at doses of 5.3–5.8 log₁₀ PFU/mouse. Three weeks later, the mice were challenged with the Ross strain of CHIKV IN at a dose 6.5 log₁₀ PFU.