Antigenic Variation of East/Central/South African and Asian Chikungunya Virus Genotypes in Neutralization by Immune Sera

<div><p>Background</p><p>Chikungunya virus (CHIKV) is a re-emerging mosquito-borne virus which causes epidemics of fever, severe joint pain and rash. Between 2005 and 2010, the East/Central/South African (ECSA) genotype was responsible for global explosive outbreaks across India, the Indian Ocean and Southeast Asia. From late 2013, Asian genotype CHIKV has caused outbreaks in the Americas. The characteristics of cross-antibody efficacy and epitopes are poorly understood.</p><p>Methodology/Principal Findings</p><p>We characterized human immune sera collected during two independent outbreaks in Malaysia of the Asian genotype in 2006 and the ECSA genotype in 2008–2010. Neutralizing capacity was analyzed against representative clinical isolates as well as viruses rescued from infectious clones of ECSA and Asian CHIKV. Using whole virus antigen and recombinant E1 and E2 envelope glycoproteins, we further investigated antibody binding sites, epitopes, and antibody titers. Both ECSA and Asian sera demonstrated stronger neutralizing capacity against the ECSA genotype, which corresponded to strong epitope-antibody interaction. ECSA serum targeted conformational epitope sites in the E1-E2 glycoprotein, and E1-E211K, E2-I2T, E2-H5N, E2-G118S and E2-S194G are key amino acids that enhance cross-neutralizing efficacy. As for Asian serum, the antibodies targeting E2 glycoprotein correlated with neutralizing efficacy, and I2T, H5N, G118S and S194G altered and improved the neutralization profile. Rabbit polyclonal antibody against the N-terminal linear neutralizing epitope from the ECSA sequence has reduced binding capacity and neutralization efficacy against Asian CHIKV. These findings imply that the choice of vaccine strain may impact cross-protection against different genotypes.</p><p>Conclusion/Significance</p><p>Immune serum from humans infected with CHIKV of either ECSA or Asian genotypes showed differences in binding and neutralization characteristics. These findings have implications for the continued outbreaks of co-circulating CHIKV genotypes and effective design of vaccines and diagnostic serological assays.</p></div

Sequences of identified B cell epitopes on the E2 glycoprotein of MY/08/065.

<p>Sequences of identified B cell epitopes on the E2 glycoprotein of MY/08/065.</p

E2-I2T, H5N, G118S and S194G substitutions within linear neutralizing epitopes enhance the neutralization activity of ECSA and Asian sera.

<p>(A) Overlapping synthetic peptides covering the E2 glycoprotein of MY/08/065 and its domains from amino acids 1–362 were screened with CHIKV immune sera at 1:1000 dilution. The black solid line represents the mean OD value of healthy controls and the dotted line represents the cut-off value (mean+3SD). The average results from 2 independent experiments are presented. In the event of two adjacent positively-mapping peptides, the peptide with the highest OD reading was taken. Key positive mapping peptides are color-coded. (B) Selected synthetic peptides were re-screened with pooled ECSA sera at lower dilutions of 1:500 and 1:250, in tetraplicate. The black solid line represents the mean OD value of healthy controls and the dotted line represents the cut-off value (mean+3SD). Key positive mapping peptides are color-coded. (C) Schematic diagram of the E2 protein showing the positions of the color-coded mapped epitopes. The numbers refer to the amino acid positions demarcating the E2 domains. N, N-link; C.arch, central arch. (D) Schematic representation of the E2 glycoprotein with the numbers indicating the amino acid positions of the glycoprotein and its domain proteins. The amino acid differences between E2 glycoproteins of ECSA (MY/08/065, ICRES1) and Asian (MY/06/37348, CAR) strains were tabulated and mapped (from amino acids 1–362). Amino acid differences within a genotype are underlined. Amino acid changes which fall within the identified linear epitopes are color-coded. (E) Immunoblotting was performed against recombinant E2 glycoproteins under reducing conditions, with each named amino acid change from the Asian to the ECSA sequence introduced independently. Mouse anti-His antibody was used as a control. (F) Seroneutralization was performed against different constructs with the CAR-2SG-ZsGreen backbone, which were rescued from the corresponding CHIKV icDNAs. Data are represented as means ± SD from 4 independent experiments at a serum dilution of 1:800 (pooled sera). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, Mann-Whitney U test. (G) Competitive peptide blocking assay was performed at 1:100 dilution with either pooled ECSA or Asian sera against ICRES1 at an MOI of 1. Data are expressed as percentages of infectivity of an infection control, and are presented as means ± SD from 2 independent experiments. *<i>P</i><0.05, **<i>P</i>< 0.01, Mann-Whitney U test, relative to unblocked condition. (H) The color-coded mapped neutralizing epitopes are localized on the structural E1-E2 heterodimer complex (based on PDB 3J2W). The epitope sequence of LP1 is only partially localized as the 3D structure is not fully resolved.</p

The E1-E211K amino acid change enhances neutralization activity of ECSA serum.

<p>(A) Schematic diagram showing the generation of fusion recombinant E2 (amino acids 1–362) and recombinant E1 (amino acids 1–412) with a 16 residue linker which has glycine/ serine spacers and octa-histidine sequence. The rE2 and rE1 in each fusion protein are from either MY/08/065 (ECSA) or MY/06/37348 (Asian) virus isolates. (B) Immunoblotting was performed under non-reducing condition. Mouse anti-E2 and mouse anti-His monoclonal antibodies were used as controls. (C) The relative binding capacity of ECSA and Asian sera (1:1000 dilution) with the fusion E2-E1 proteins were determined in ELISA as (OD samples/mean OD samples tested with rE2-E1-ECSA) × 100. Data are presented as means ± SD (<i>n</i> = 4). *<i>P</i><0.05, Mann-Whitney U test. (D) Schematic representation of the E1 glycoprotein with the numbers indicating the amino acid positions of the glycoprotein and its domain proteins. The amino acid differences between E1 glycoprotein of ECSA (MY/08/065, ICRES1) and Asian (MY/06/37348, CAR) were tabulated and mapped (from amino acids 1–412). Amino acid differences within a genotype are underlined. FL, fusion loop; C. tail, cytoplasmic tail. (E) Immunoblotting was performed against fusion E2-E1 glycoprotein under non-reducing conditions, with each named amino acid change from the Asian to the ECSA sequence introduced independently. Mouse anti-His antibody was used as a control. (F) Seroneutralization was performed against different constructs carrying indicated mutations in the CAR-2SG-ZsGreen backbone, which were rescued from the corresponding icDNA clones of CHIKV. Data are represented as means ± SD from 4 independent experiments at a serum dilution of 1:800 (pooled sera). *<i>P</i><0.05, ***<i>P</i><0.001, Mann-Whitney U test, relative to CAR. (G) Seroneutralization was performed against the CAR-E1-E211K rescued virus at a serum dilution of 1:800 with 23 individual serum samples. ***<i>P</i><0.0001, Wilcoxon matched-pairs signed rank test. (H) The amino acid position of K211 which affects neutralization activity is localized on the structural E1-E2 heterodimer complex (based on PDB 3J2W).</p

Neutralizing antibodies of immune sera interact with the epitopes on E2 and E1-E2 glycoproteins.

<p>(A) CHIKV antibody titer against recombinant E1 glycoprotein (100 ng) was determined in ELISA. The ELISA was performed at different serum dilutions using pooled sera. The dotted line represents the cut-off value (mean + 3SD) derived from healthy controls. (B) Competitive blocking assay was performed at 1:200 dilution in triplicate using 7 pools of ECSA and Asian sera, with similar neutralizing titers in each pool. Data are expressed as percentages of infectivity of an infection control, and are presented as means ± SEM. ***<i>P</i>< 0.001, repeated measures ANOVA with the Bonferroni multiple comparison test. (C) Schematic diagram showing the construction of chimera viruses with replacement of E2 or E1/E2 from SFV into the CHIKV ICRES1 backbone. Seroneutralization was performed against the chimera constructs and the percentage of infectivity was compared to that obtained with ICRES1. Data are represented as means ± SD from 4 independent experiments at a serum dilution of 1:100 (pooled sera). ***<i>P</i><0.01, Kruskal-Wallis test. G, genomic promoter; SG, subgenomic promoter.</p

Differential neutralization capacity and antibody binding properties of immune sera against ECSA and Asian CHIKV.

<p>(A) Sera collected from ECSA and Asian CHIKV outbreaks have differential neutralizing capacity against MY/08/065 (ECSA) and MY/06/37348 (Asian) isolates of CHIKV. Results are expressed as a percentage of virus control. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, two-way ANOVA with the Bonferroni multiple comparisons test. Data are presented as means ± SEM from 23 (ECSA) and 40 (Asian) individual serum samples. (B) Neutralization titers (NT₅₀) of DTT-treated sera were determined by non-linear regression fitting. ***<i>P</i><0.001, Wilcoxon matched-pairs signed rank test. (C) ECSA and Asian sera were cross-screened against both CHIKV isolates (10⁵ pfu, treated with 1% Triton X-100) in ELISA at different serum dilutions. Data are presented as means ± SEM from 23 (ECSA) and 40 (Asian) individual serum samples. **<i>P</i><0.01, ***<i>P</i><0.001, two-way ANOVA with the Bonferroni multiple comparisons test. (D) Seroneutralization was performed against different strains of CHIKV, ICRES1 (ECSA) and CAR (Asian), which were rescued from icDNA CHIKV, at a serum dilution of 1:800. **<i>P</i><0.01, ***<i>P</i><0.0001, Wilcoxon matched-pairs signed rank test. (E) Immunoblotting was performed under non-reducing and reducing conditions against rE2 and CHIKV from MY/08/065 and MY/06/37348. Mouse anti-His was used as a control and pooled sera were diluted at 1:1000. (F) Immunoblotting was performed under non-reducing and reducing conditions against ICRES1 and CAR. Mouse anti-E2 was used as a control and pooled sera were diluted at 1:1000. (G) Antibody titers of CHIKV immune sera (IgG) were quantified by end-point titer ELISA using whole virus antigen and recombinant E2 (rE2) derived from MY/08/065. Middle line, median; plus sign, mean; upper and lower boundaries of the box, inter-quartile range; whiskers, range of values.</p

Sequence variation of a linear neutralizing epitope influences the spectrum of cross-neutralization across genotypes.

<p>(A) Synthetic peptides LP1A and LP1 were screened with ECSA immune sera at 1:500 dilution in tetraplicate. The dotted line represents the cut-off value (mean + 3SD). *<i>P</i><0.05, Mann-Whitney U test. (B) Recombinant viruses were pre-treated with 1% Triton X-100, then coated at 5 x 10⁵ pfu per well, and a binding assay was performed with 1μg/ml of antibody. The seroneutralization assay was performed with infection at an MOI of 50 against 25μg/ml of antibody. Data are represented as means ± SD from 3 independent experiments. ***<i>P</i><0.001, repeated measures ANOVA with the Bonferroni multiple comparisons test. (C) Synthetic peptides LP1A and LP1 were screened with Asian immune sera at 1:500 dilution in tetraplicate. The dotted line represents the cut-off value (mean + 3SD). *<i>P</i><0.05, Mann-Whitney U test. (D) Seroneutralization of anti-LP1A against CAR and CAR-E2-I2T-H5N at an antibody concentration of 25 μg/ml. Data are represented as means ± SD from 3 independent experiments.(E) Competitive peptide blocking assay was performed at 1:100 dilution with either LP1A or LP1 in pooled Asian sera against ICRES1 at an MOI of 1. Data are expressed as percentages of infectivity of an infection control, and are presented as means ± SD (<i>n</i> = 6). **<i>P</i>< 0.01, Mann-Whitney U test, relative to unblocked condition. (F) Structural images illustrating the changes of surface electrostatic potential due to differences in amino acid positions 2 and 5 within LP1A (Asian) and LP1 (ECSA) sequences (red arrows).</p

Anti-CHIKV IgM provides a short period of protection up to day 10 during early phase of infection.

<p>(A) Anti-CHIKV IgM and IgG were measured by ELISA. Samples were categorized by time from disease onset. **<i>P</i><0.01, ***<i>P</i><0.001, Mann-Whitney U test compared to healthy controls. The dotted lines represent the cut-off value. All the samples were within the detection limit except one. (B) The neutralizing titers of IgM, IgG and total antibodies were plotted by days from disease onset. Each point indicates the median of NT₅₀. (C) Ratios of NT₅₀ IgM over NT₅₀ IgG were calculated and plotted for serum samples by times from disease onset (panel B). A ratio of 1 indicates equal strength of NT₅₀ IgM over NT₅₀ IgG. (D) Representative immunofluorescent microscopic images of CHIKV-infected cells incubated with 1:100 diluted serum under different treatment conditions. Objective magnification: 5× from 1 field of view. The serum sample used was from panel B1, collected 4 days post-onset of illness, with IgM and IgG titers of 20480 and 4096, respectively. (E) Panel B1 was divided into groups with either high or low NT₅₀ IgG. Neutralization of virus infectivity due to IgM or IgG was compared to neutralizing capacity due to total antibodies. Results are expressed as percentage of virus control. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 by two-way ANOVA with Bonferroni multiple comparisons test. Data are presented as mean ± SD for individual samples with low NT₅₀ IgG and mean ± SEM for 12 samples with high NT₅₀ IgG. (F) Panel B1 was divided into groups with either high or low NT₅₀ IgG ratio. IgG titers against CHIKV and rE2 were measured. *<i>P</i><0.05, Mann-Whitney U test. The lines in the middle of the boxes indicate medians; the upper and lower boundaries of the box indicate inter-quartile ranges; and the whiskers indicate ranges of values.</p

Anti-CHIKV IgM preferably targets epitopes on E1-E2 glycoproteins.

<p>(A) IgM immunoblotting was performed using approximately 1 μg of recombinant protein (E1 and E2) and 2.5 μg of whole virus antigen under non-reducing conditions at serum dilutions of 1:100 and 1:400 using pooled serum samples from panel B1. Mouse anti-His (α-His) was used as a control. (B) IgM reactivity against rE1 and rE2 in capture ELISA was investigated. The experiments were performed at 1:100 serum dilution. The dotted lines represent the cut-off value. (C) Immunoblotting was performed under non-reducing and reducing conditions at a serum dilution of 1:400 against fusion recombinant E2 and recombinant E1 glycoprotein (rE2-E1-ECSA). (D) Schematic diagram showing the chimeras used in seroneutralization with comparison of infectivity using pooled serum samples from panel B1. Data are presented as means ± SD from 4 independent experiments at a serum dilution of 1:100. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, Kruskal-Wallis test.</p

The appearance of CHIKV-specific neutralizing antibodies is associated with a reduction of viremia.

<p>(A) Seroneutralization was performed on sera from panel A which had known viral loads. Experiments were performed in triplicate at 1:100 dilution for detection of neutralizing IgM and IgG. The dotted lines represent the cut-off value determined from the mean—SD values from healthy control sera. (B) Neutralization titers (NT₅₀) of total antibodies and previously-determined viral loads [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171989#pone.0171989.ref026" target="_blank">26</a>] of serum samples (panel A) collected at different times of disease onset were plotted. Each point indicates the median of RNA copies/ml targeting E1 and NT₅₀, with the inter-quartile range. The dashed line indicates the limit of quantification (1 log₁₀ RNA copies/reaction or 4.76 log₁₀ RNA/ml) of the E1 positive-strand PCR assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171989#pone.0171989.ref026" target="_blank">26</a>].</p