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. 2013 Jun 12;8(6):e66507.
doi: 10.1371/journal.pone.0066507. Print 2013.

Antibody responses in humans infected with newly emerging strains of West Nile Virus in Europe

Stefan Chabierski  1 , Gustavo R Makert, Alexandra Kerzhner, Luisa Barzon, Petra Fiebig, Uwe G Liebert, Anna Papa, Justin M Richner, Matthias Niedrig, Michael S Diamond, Giorgio Palù, Sebastian Ulbert
Affiliations

Antibody responses in humans infected with newly emerging strains of West Nile Virus in Europe

Stefan Chabierski et al. PLoS One. .

Abstract

Infection with West Nile Virus (WNV) affects an increasing number of countries worldwide. Although most human infections result in no or mild flu-like symptoms, the elderly and those with a weakened immune system are at higher risk for developing severe neurological disease. Since its introduction into North America in 1999, WNV has spread across the continental United States and caused annual outbreaks with a total of 36,000 documented clinical cases and ∼1,500 deaths. In recent years, outbreaks of neuroinvasive disease also have been reported in Europe. The WNV strains isolated during these outbreaks differ from those in North America, as sequencing has revealed that distinct phylogenetic lineages of WNV concurrently circulate in Europe, which has potential implications for the development of vaccines, therapeutics, and diagnostic tests. Here, we studied the human antibody response to European WNV strains responsible for outbreaks in Italy and Greece in 2010, caused by lineage 1 and 2 strains, respectively. The WNV structural proteins were expressed as a series of overlapping fragments fused to a carrier-protein, and binding of IgG in sera from infected persons was analyzed. The results demonstrate that, although the humoral immune response to WNV in humans is heterogeneous, several dominant peptides are recognized.

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Conflict of interest statement

Competing Interests: The Fraunhofer Institute of Cell Therapy and Immunology has filed a patent application on a diagnostic test for WNV infections, which includes some of the fusion proteins described ("methods for the specific detection of antibodies against flaviviruses"; Appl. Number to be assigned). This does not alter the adherence of the authors to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Purification and testing of recombinant proteins.
A: SDS-PAGE showing crude lysates of protein-expressing bacteria and purification steps. M, size marker; Lane 1, culture expressing GST (arrowhead); lane 2, culture expressing peptide C41–70 (asterisk); lane 3, peptide C41–70 after glutathione-affinity-purification; lane 4, peptide C41–70 after gelfiltration. B: ELISA using serum I3 on GST purified only with glutathione sepharose (white column, 500 ng) or additionally with gel filtration chromatography (black column, 2000 ng). Values are the mean of two independent experiments (performed in duplicate), error bars represent the standard deviation. Statistical analysis to evaluate the difference between the two signals was performed by using an unpaired t-test, (asterisks, p<0,001).
Figure 2
Figure 2. Optimizing antibody binding to selected peptides.
ELISA plates were coated with increasing amounts of the indicated peptides fused to GST and incubated with human sera: WNV positive sera I2, I4; G11, G12, G15 and negative serum N5. Values are the mean of two independent experiments (performed in duplicate), error bars represent the standard deviation.
Figure 3
Figure 3. Analysis of the binding property of selected recombinant peptides with human sera in an ELISA.
30-mer peptides spanning the WNV-proteins capsid, prM/M and E, fused to GST, were incubated with human sera from outbreaks in Italy (I1-8), Greece (G1–G15) and USA (US1-2). Those peptides displaying signals with an OD >0,5 are shown (the total number of peptides is shown in Figure S1).Values represent the absorption over cut-off (mean of four negative sera plus two standard deviations) and are derived from at least two independent experiments (performed in duplicate). Error bars represent the standard deviation. The background (binding of the serum to GST) was subtracted.
Figure 4
Figure 4. Analysis of binding properties of human sera with recombinant E ectodomain in an ELISA.
Sera used in this study were incubated with the recombinant E-ectodomain protein (lineage 1). Mean values of two independent experiments (performed in duplicate) are shown, and error bars represent the standard deviation.
Figure 5
Figure 5. Competition of antibody binding between selected peptides and recombinant E-ectodomain.
The indicated sera were pre-incubated with recombinant peptides E71–100, E231–260, E371–400 (all fused to GST) or GST alone, before they were bound to recombinant E-ectodomain of WNV. Values are derived from at least two independent experiments (performed in duplicate). Error bars represent the standard deviation. Statistical analysis to evaluate the difference between the signals of the competitor peptide and the control competitor (GST alone) were performed by using a Mann-Whitney Rank Sum Test (asterisks, p<0,05).
Figure 6
Figure 6. Structure of the E-ectodomain of WNV.
The sequences corresponding to the peptides E71–100, E231–260 and E371–400 are highlighted in color. DI, DII and DIII indicate the respective domains.
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Grants and funding

This work was supported by the European Commission under FP7, project number 261426 (WINGS; Epidemiology, Diagnosis and Prevention of West Nile Virus in Europe). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.