Attenuation of Chikungunya virus vaccine strain 181/clone 25 is determined by two amino acid substitutions in the E2 envelope glycoprotein

Abstract

Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis-acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies.

Fig 1

Schematic representation of the CHIKV genome. (a) Positions of nonsynonymous mutations in vaccine strain 181/25. (b) Mutations cloned into the CHIKV LR genomic backbone. Numbers correspond to amino acid positions of the mutations in the respective viral protein. SG, subgenomic.

Fig 2

Mean body weights of 6- to 7-day-old CD1 mice (n = 10) after SC infection with 10⁵ PFU of CHIKV strain LR, mutants, or vaccine strain 181/25.

Fig 3

Survival of 8- to 10-week-old A129 mice (n = 4 or 5) after ID infection with 10⁴ PFU of CHIKV strain LR, mutants, or vaccine strain 181/25. Animals were monitored for 18 days.

Fig 4

Morbidity in 8- to 10-week-old A129 mice (n = 4 or 5) after ID infection with 10⁴ PFU of CHIKV strain LR, mutants, or vaccine strain 181/25. (a) Body weight changes. (b) Body temperature changes. (c) Footpad swelling at day 2 postinfection. Asterisks indicate P values (determined by one-way ANOVA with a Tukey-Kramer posttest) of differences from 181/25 or PBS control groups. *, P < 0.05; **, P < 0.01.

Fig 5

Schematic representations of CHIKV AF15561 genomes with cloned mutations. Numbers correspond to amino acid positions of the mutations in the respective viral proteins.

Fig 6

Survival of 5- to 8-week-old A129 mice (n = 9) after ID infection with 10⁴ PFU of CHIKV strain AF15561, mutants, or vaccine strain 181/25. Animals were monitored for 14 days.

Fig 7

Morbidity in 5- to 8-week-old A129 mice (n = 9) after ID infection with 10⁴ PFU of CHIKV strain AF15561, mutants, or vaccine strain 181/25. (a) Body weight changes. (b) Body temperature changes. (c) Footpad swelling. ***, P < 0.001 (determined by one-way ANOVA with a Tukey-Kramer posttest) compared to the 181/25 group. p.i., postinfection.

Fig 8

Crystal structure of the mature envelope glycoprotein complex of CHIKV fitted into a cryoelectron microscopic reconstruction map of Western equine encephalitis virus glycoprotein spikes on the surface of the viral particle (39), prepared using UCSF Chimera software (30). (a) Cryoelectron microscopic map of Western equine encephalitis virus. (b) Top view of the three E2-E1 heterodimers forming the spike, magnified from a circle in panel a, with the CHIKV E2-E1 crystal structure fitted into one of the heterodimers. (c) Same spike with the CHIKV crystal structure as in panel b, rotated 90°. (d) Ribbon diagram of the CHIKV heterodimer (Molecular Modeling Database identification no. 86612, chains B and F [52]) in the same orientation as in panel b. E2 is yellow, and E1 is blue. E2 amino acid positions T12 and G82 are shown as red spheres. E2 positions 76 and 114 are residues described in the literature as responsible for heparan sulfate binding in SINV (4, 19) and are shown as magenta and cyan spheres, respectively.