Linear B-Cell Epitopes in Human Norovirus GII.4 Capsid Protein Elicit Blockade Antibodies

Abstract

Human norovirus (HuNoV) is the leading cause of nonbacterial gastroenteritis worldwide with the GII.4 genotype accounting for over 80% of infections. The major capsid protein of GII.4 variants is evolving rapidly, resulting in new epidemic variants with altered antigenic potentials that must be considered for the development of an effective vaccine. In this study, we identify and characterize linear blockade B-cell epitopes in HuNoV GII.4. Five unique linear B-cell epitopes, namely P2A, P2B, P2C, P2D, and P2E, were predicted on the surface-exposed regions of the capsid protein. Evolving of the surface-exposed epitopes over time was found to correlate with the emergence of new GII.4 outbreak variants. Molecular dynamic simulation (MD) analysis and molecular docking revealed that amino acid substitutions in the putative epitopes P2B, P2C, and P2D could be associated with immune escape and the appearance of new GII.4 variants by affecting solvent accessibility and flexibility of the antigenic sites and histo-blood group antigens (HBAG) binding. Testing the synthetic peptides in wild-type mice, epitopes P2B (336-355), P2C (367-384), and P2D (390-400) were recognized as GII.4-specific linear blockade epitopes with the blocking rate of 68, 55 and 28%, respectively. Blocking rate was found to increase to 80% using the pooled serum of epitopes P2B and P2C. These data provide a strategy for expanding the broad blockade potential of vaccines for prevention of NoV infection.

Keywords: GII.4 genotype; linear blockade epitope; molecular dynamic simulation; norovirus.

Figure 1

Predicted B-cell epitopes in NoV GII.4 variants and their evolutionary pattern. (A) Linear B-cell epitopes were predicted within norovirus Sydney GII.4 capsid protein using IEDB, Epitopia27 and BepiPred prediction tools: epitopes with antigenicity index higher that 0.4 were considered as antigen. NoV crystal structure (1IHM) was taken from protein data bank and structure was generated with chimera tool. (B) Evolutionary pattern of the predicted linear and conformation epitopes was determined within the GII4 variants connected with norovirus outbreaks. Yellow and green colors have been used to differentiate closed neighboring conformational epitopes (C) Columns of heterogenecity within P2 domain. Respective variants constituted yearly consensus sequences till they were replaced by the next variant. Positively selected sites are labelled with an asterisk below the columns.

Figure 2

Surface accessibility of amino acid residues within epitopes sites. Capsid protein from norovirus variants responsible for outbreaks were assessed for changes in surface accessibility within epitopes sites using the Emini Surface Accessibility Prediction tool. (A) Epitopes within the P2 domain showed changes in a mutation-related manner. HBGA binding sites are located within epitopes P2B, P2C and P2D; (B) Epitope sites in the surface exposed sites of P1 domain (P1B and P1C) were stable over years.

Figure 3

(A) Predicted secondary structure for norovirus GII.4 capsid protein. Yellow arrows represent β-strands. Amino acid substitution in variable sites caused notable changes in the P2 domain ß-strands. (B) Structural location of putative epitopes in GII.4 Sydney 2012 used in the present study.

Figure 4

Molecular dynamic simulation analysis of NoV GII.4 P2 domain. Homology 3-D models were generated, protein models were visualized and after validation analysis were subjected to MD simulation. (A) RMSD analysis showed distinct patterns of instability in the epitopes sites within the P2 domain of GII.4 variants. (B) RMSF analysis revealed high fluctuation in epitope sites. Black and red color denotes low and high RMSF values.

Figure 5

Molecular docking of NoV capsid protein and HBGA. (A) Predicted HBGA binding sites. Comparing GII.4 variants, two HBGA binding patterns were predicted; each GII.4 variant showed similar binding pattern for the both Lewis^a and Lewis^b saccharides. (B) 3-D protein structure models of GII.4 capsid proteins from 1987, 2004, 2006 and 2012 are represented along docked pose (zoomed plot) of HBGA with P2 domains.

Figure 6

Characterization of predicted epitopes P2A-E within NoV GII.4 capsid protein. NoV GII.4 Sydney 2012 VLPs were produced using baculovirus expression system: (A) NoV VLPs were purified on sucrose gradient; (B) negative-stained VLPs were visualized by electron microscopy; (C) VLPs were tested with Western blotting. (D) BALB/c mice were immunized with synthetic epitopes three times; two weeks after the last immunization, serum antibody level was determined by ELISA (E) and blockade of NoV GII.4 Sydney 2012 VLPs binding to HBGA H-type-3 receptor by sera of the immunized mice was tested (F): Blocking activities were determined for pooled sera from five mice in each group. PBS served as negative control.

Figure 7

Norovirus capsid protein surface loops. Red indicates P2 domain, while blue and green indicate P1 and S domain. The surface loops that are suitable sites for insertion of foreign antigen are shown in white.