Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus

Abstract

Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

Figure 1. Map of Gabonese villages where the 1996 and 2001 outbreaks occurred.

Figure 2. Mean OD values at 1∶400 serum dilution in patients infected with EBOV during three outbreaks in Gabon, 7 days after symptom onset (Day 7, early humoral response) and 7 or 11 years later (2008, late humoral response), and also in anti-EBOV IgG+ asymptomatic individuals who had never had clinical signs of hemorrhagic fever or who lived in non epidemic areas*.

ODs are color-coded to indicate their intensity (yellow to red). The different colored regions in the proteins indicate the immunodominant domains identified here. *Blank cells indicate that the mean OD was lower than the cut-off. OD: Optical Density. The absorbance cut-off used to identify reactive epitopes is described in the Methodology section.

Figure 3. Epitopes reacting with sera collected from survivors of the three outbreaks in Gabon, 7 days after symptom onset, and from asymptomatic individuals (Asympt.) who had never had clinical signs of hemorrhagic fever or who lived in non epidemic areas, are indicated in color.

GP epitopes shown in different colors correspond to the regions of the GP protein structure in Figure 4. Epitopes reacting with sera from survivors of the three outbreaks, collected 7 or 11 years after recovery, are indicated in italics. The principal immunogenic regions are underlined.

Figure 4. Crystal structure of the trimeric prefusion EBOV GP viewed from the top (above) and side (below) .

The three monomers in the complex are colored different shades of grey. Immunodominant regions identified in GP are indicated in color for survivors and asymptomatic patients. The color code matches that of the epitopes identified in Figure 3. Molecular surface of the GP trimer viewed from the side (left) and top (right), as viewed down the three-fold axis. Protein Data Bank file number: 3CSY.