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. 2015 Jan 8;9(1):e3445.
doi: 10.1371/journal.pntd.0003445. eCollection 2015 Jan.

Sero-prevalence and cross-reactivity of chikungunya virus specific anti-E2EP3 antibodies in arbovirus-infected patients

Affiliations

Sero-prevalence and cross-reactivity of chikungunya virus specific anti-E2EP3 antibodies in arbovirus-infected patients

Yiu-Wing Kam et al. PLoS Negl Trop Dis. .

Abstract

Chikungunya virus (CHIKV) and clinically-related arboviruses cause large epidemics with serious economic and social impact. As clinical symptoms of CHIKV infections are similar to several flavivirus infections, good detection methods to identify CHIKV infection are desired for improved treatment and clinical management. The strength of anti-E2EP3 antibody responses was explored in a longitudinal study on 38 CHIKV-infected patients. We compared their anti-E2EP3 responses with those of patients infected with non-CHIKV alphaviruses, or flaviviruses. E2EP3 cross-reactive samples from patients infected with non-CHIKV viruses were further analyzed with an in vitro CHIKV neutralization assay. CHIKV-specific anti-E2EP3 antibody responses were detected in 72% to 100% of patients. Serum samples from patients infected with other non-CHIKV alphaviruses were cross-reactive to E2EP3. Interestingly, some of these antibodies demonstrated clearly in vitro CHIKV neutralizing activity. Contrastingly, serum samples from flaviviruses-infected patients showed a low level of cross-reactivity against E2EP3. Using CHIKV E2EP3 as a serology marker not only allows early detection of CHIKV specific antibodies, but would also allow the differentiation between CHIKV infections and flavivirus infections with 93% accuracy, thereby allowing precise acute febrile diagnosis and improving clinical management in regions newly suffering from CHIKV outbreaks including the Americas.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Antibody profiles of CHIKV-infected patients.
A, Schematic diagram on the classification of clinical specimens. Four hundred and ten clinical specimens were included in this study, 339 samples have validated by in-house screening methods including 60 flaviviruses-positive samples and 279 alphaviruses-positive samples. Validated samples were further sub-categorized into 4 groups: CHIKV; Non-CHIKV (positive for alphavirus); DENV; and non-DENV (flavivirus-positive). Another 71 samples of febrile patients with unknown cause of infection were also included in this study. B, CHIKV E2EP3-specific IgG antibody detection in patients' serum samples (total 38 patients, n = 260) at a dilution of 1∶4000 were determined by peptide-based ELISA. Black solid line represents the mean value of the healthy donors and dotted line represents the value of mean ±3 SD. Values above mean ±3 SD are considered positive anti-E2EP3 IgG antibody response. Data are presented as mean. Patient samples were categorized into different phase including acute (1–6 days); early convalescent (7–13 days); late convalescent (14–29 days); chronic (30–176 days). C, Pie-chart shows the percentage of patients with positive or negative anti-E2EP3 IgG antibody response (upper panel), and the percentage of patients with positive or negative anti-CHIKV IgG response using immunofluorescence-based biochips (CHIKV IIFT, EUROIMMUN AG) (lower panel).
Figure 2
Figure 2. Cross-reactivity of anti-alphaviruses and anti-flaviviruses antibodies with CHIKV E2EP3.
Patient serum samples caused by non-CHIKV alphaviruses infections (n = 19) and flaviviruses infections (n = 60) were screened for cross-reactivity against CHIKV E2EP3 by peptide-based ELISA at a dilution of 1∶4000. A, Pie-chart shows the percentage of non-CHIKV alphaviruses-infected patients samples with positive or negative anti-E2EP3 antibody response. B, Pie-chart shows the percentage of flaviviruses-infected patient samples with positive or negative anti-E2EP3 antibody response. C - D, Pie-chart shows the percentage of DENV-infected and non-DENV flaviviruses-infected patient samples with positive or negative anti-E2EP3 IgG antibody response respectively. E, Detection of CHIKV E2EP3 by serum samples of unknown infections. Serum samples (n = 71) of febrile patients from unknown cause of infections were screened by peptide-based ELISA at a dilution of 1∶4000. Pie-chart shows the percentage of patients' samples with positive or negative anti-E2EP3 antibody response. F, Bar-chart shows the number of non-CHIKV alphaviruses-infected, E2EP3 cross-reactive samples with and without CHIKV neutralizing activity against the two CHIKV isolates (CHIKV (A226) and CHIKV (A226V)).
Figure 3
Figure 3. Association of E2EP3 cross-reactive antibody responses with CHIKV neutralizing activity.
A, Anti-E2EP3 antibody response in serum samples (High E2EP3 IgG responders, n = 10; Low E2EP3 IgG responders, n = 15), at a dilution of 1∶4,000 were determined by ELISA using biotinylated E2EP3 peptides. Data are presented as mean ± SEM. Data are representative of two independent experiments with similar results. Statistical significance was measured using Mann-Whitney U test (***P<0.0001). B, In-vitro neutralizing activity against CHIKV in High and Low E2EP3 IgG responders. Histogram shows the percentage of patient samples with or without CHIKV neutralizing activity. Statistical significant was measured using 2-sided Fisher exact test between the patient samples with and without CHIKV neutralizing activity in the 2 responder groups. *P = 0.028. C - D, E2EP3 sequence from CHIKV was aligned to corresponding sequences from 9 non-CHIKV alphaviruses (ONNV, RRV, SFV, MAYV, BFV, SINV, VEEV, EEEV and WEEV). Conserved amino acid (A.A.) are indicated by a *. Percentage similarity between CHIKV E2EP3 sequence and corresponding sequence from each non-CHIKV alphavirus were calculated with SIAS (http://imed.med.ucm.es/Tools/sias.html). Percentage similarity relative to CHIKV E2EP3 is indicated by a bar-chart. Viruses classified as Old World alphaviruses are marked by the grey shading box, while viruses classified as New World alphaviruses are marked by the white box. NCBI Genbank accession numbers of ten selected alphaviruses (CHIKV - DQ443544, ONNV - O90369, RRV - AEC49728, SFV - ABA29033, MAYV - AAL79764, BFV - NP819000, SINV - ACU25462, VEEV - AAB02519, EEEV - NP740646, WEEV - NP818940).

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