Figure 1.
Reduced neutralizing potency of mAb 5F10 and 8B10 against CHIKVs amplified under selective pressure.
CHIK/Irr, CHIK/5F, CHIK/8B and CHIK/5F+8B indicate CHIKV rescued from 8 serial cell passages under the continuous mAb pressure of Irr.IgG1, 5F10, 8B10 and 5F10+8B10, respectively. (A) The neutralizing potency of CHIKV-specific mAb was evaluated in PRNT over a concentration range of 1 ng-100 µg/ml. Displayed are the mean and SEM from 3 independent experiments performed in duplicate, with non-linear regression fitting curves. Regression could not be calculated for CHIK/5F due to non convergence. (B) The extrapolated IC50 from 2 or 3 independent experiments are shown, alongside the mean and SEM.
Table 1.
Amino acid variations in the E1 and E2 glycoproteins among CHIKV variants amplified over 8 rounds under mAb pressure.
Table 2.
Next Generation Sequencing of non-clonal CHIK/Irr after 8 rounds under mAb pressure.
Table 3.
Amino acid variations in the E1 and E2 glycoproteins among clonal CHIKVs.
Figure 2.
5F10 and 8B10 mAb neutralizing potency against clonal CHIKVs.
CHIKV variants have been labelled according to their residue substitutions and corresponding mAb added to the culture medium during the serial passages. (A) Neutralizing capacities of the mAb against plaque-purified CHIKV were evaluated in PRNT over a concentration range of 1 ng-100 µg/ml. Shown are the mean and SEM from 4 independent experiments performed in duplicate with non-linear regression fitting curves. (B) The calculated IC50 from 3 or 4 independent experiments and mean ± SEM are shown (P-values were calculated using Kruskal-Wallis test and Dunn's post-test, and compare with the IC50 against CHIKwt: *, p<0.05; ***, p<0.001).
Figure 3.
Analysis of 5F10 and 8B10 mAb binding to clonal CHIKVs.
(A) Analysis of mAb binding to CHIKV-infected cells by immunofluorescence assay. HEK293T cells either non-infected, or infected with the indicated CHIKVs, were probed with mAb 5F10, 8B10, anti-CHIKV plasma, or with irrelevant IgG1. Images were captured at 100× magnification. (B) Quantitative analysis of mAb binding to CHIKV-infected cells by Cellomics ArrayScan. HEK293T cells, either mock infected or infected with the indicated CHIKVs, were probed with mAb 5F10, 8B10, anti-CHIKV plasma, or with irrelevant IgG1. Images were captured at 10× magnification. Displayed are mean fluorescent intensity (MFI) and SEM from 3 independent experiments performed in quadruplicate (P-values were calculated by Kruskal-Wallis test and Dunn's post-test, and compare with the MFI of CHIKwt-infected cells: *, p<0.05; **, p<0.01; ***, p<0.001). (C) ELISA analysis of mAb binding to plaque-purified CHIKV particles. ELISA plates were coated with 104 UV-inactivated CHIKV particles, prior to being incubated with mAb (0.1 ng-100 µg/mL). Bound mAb were detected using HRP-conjugated goat anti-human IgG and TMB substrate. The OD was measured at 450 nm. Shown are mean and SEM from 3 independent experiments performed in duplicate.
Figure 4.
Location of E1.101, E2.12, E2.82 and E2.216 in the CHIKV E1/E2 heterodimer.
Based on structural data retrieved from protein database records, the 3D organization of E1 (pale yellow), E2 (white), and the E1 fusion loop (pink) are shown for 3N44 under neutral pH conditions. (A) Front view of E2 with the location of E2.216 (green) and E2.82 (orange). (B) Back left view of E2 and E1 with the location of E2.12 (blue) and E1.101 (red).
Figure 5.
Location of Sindbis virus residues E1.S101 (CHIKV E1.T101) and E2.T9 (CHIKV E2.T12) in the E1/E2 heterodimer.
Indicated residues were located based on structural data retrieved from protein data base records (3MUU, under acidic pH conditions).
Figure 6.
Viral fitness of CHIKV neutralization-escape mutants.
(A) Vero cells were infected with plaque-purified CHIKV (MOI = 0.1). The number of PFU within the supernatants was determined by Plaque Assay at various times post-infection. Shown are mean and SEM from 3 independent experiments performed in duplicate. (B) Shown are typical plaque patterns by crystal violet staining at 12 h post-infection. (C) AGR129 mice were inoculated with plaque-purified CHIKV (103 PFU). PBS-inoculated mice were used as negative controls. Mice were observed every 12 h to determine post-infection survival. Shown are the survival curves derived from 3 independent experiments (CHIKV, n = 10; Mock infected, n = 6). P-values were determined by Grehan-Breslow-Wilcoxon test and compare CHIKV mutants with CHIKwt. (D) Mice were inoculated with CHIKwt or 5F+8B/E2.R82G (103 PFU) and the CHIKV load in serum and liver was quantified 48 h post-infection by TCID50. Shown are mean and SEM for 5 mice per group. P-values were determined using Mann-Whitney test (*, p<0.05). (E) At 48 h post-infection, the amount of CHIKV (-)RNA in liver was determined by quantitative RT-PCR. Shown are mean and SEM for 5 mice per group. P-values were determined using Mann-Whitney test (**, p<0.01).
Figure 7.
Quantification of CHIKV cell-to-cell transmission.
CHIKV-infected HEK293T cells (producer cells) were co-cultured with CFSE-labeled naïve HEK293T cells (target cells) in the absence or presence of mAb 8B10. (A) The number of infected cells was quantified by flow-cytometry either immediately (T = 0 h), or after 16 h of co-culture (T = 16 h). Above each panel are shown the mean number of infected producer (T = 0 h) or target (T = 16 h) cells alongside the SEM (5 independent experiments performed in duplicate). P-values were determined using Wilcoxon test, and compare CHIKwt and 5F+8B/E2.R82G infectivity (*, p<0.05). (B) After 16 h of co-culture, the number of extra-cellular CHIKV particles within the supernatants was determined by Plaque Assay. Shown are mean and SEM from 5 independent experiments performed in duplicate.
Figure 8.
CHIKV is polarized to sites of cell-cell contact under neutralizing antibody pressure.
CHIKV-infected HEK293T cells were co-cultured with CFSE-labeled naïve HEK293T cells (green) in the absence or presence of mAb 8B10. After 16 h of co-culture the cells were fixed, permeabilized, and stained for alphavirus expression (Alexa 647, red). Newly infected target cells appear orange. Magnification: ×40 or ×80.