Monoclonal antibody-based antigenic mapping of norovirus GII.4-2002

Abstract

Noroviruses are the primary cause of epidemic gastroenteritis in humans, and GII.4 strains cause ∼80% of the overall disease burden. Surrogate neutralization assays using sera and mouse monoclonal antibodies (MAbs) suggest that antigenic variation maintains GII.4 persistence in the face of herd immunity, as the emergence of new pandemic strains is accompanied by newly evolved neutralization epitopes. To potentially identify specific blockade epitopes that are likely neutralizing and evolving between pandemic strains, mice were hyperimmunized with GII.4-2002 virus-like particles (VLPs) and the resulting MAbs were characterized by biochemical and immunologic assays. All of the MAbs but one recognized GII.4 VLPs representing strains circulating from 1987 to 2009. One MAb weakly recognized GII.4-1987 and -1997 while strongly interacting with 2002 VLPs. This antibody was highly selective and effective at blocking only GII.4-2002-ligand binding. Using bioinformatic analyses, we predicted an evolving GII.4 surface epitope composed of amino acids 407, 412, and 413 and subsequently built mutant VLPs to test the impact of the epitope on MAb binding and blockade potential. Replacement of the 2002 epitope with the epitopes found in 1987 or 2006 strains either reduced or ablated enzyme immunoassay recognition by the GII.4-2002-specific blockade MAb. These data identify a novel, evolving blockade epitope that may be associated with protective immunity, providing further support for the hypotheses that GII.4 norovirus evolution results in antigenic variation that allows the virus to escape from protective herd immunity, resulting in new epidemic strains.

Fig 1

EIA cross-reactivity of anti-GII.4-2002 MAbs to NoV VLPs. Purified MAbs were assayed by EIA for reactivity to GI and GII NoV VLPs. (A) MAbs with cross-genogroup reactivity. (B) MAbs with cross-genotype reactivity. Bars represent means with standard errors. Positive reactivity was defined as a mean optical density (OD) at 405 nm ≥3 time that of the background (dashed line). Asterisks indicate VLPs with reactivity significantly different from that of GII.4-2002.

Fig 2

Characterization of PGM HBGA expression and VLP binding. (A) The HBGA phenotype of commercial PGM was determined by reactivity to a panel of anti-HBGA MAbs by EIA. Asterisks indicate HBGAs with reactivity significantly different from that of B antigen. (B) Binding to synthetic HBGA found in PGM of the GII.4 VLPs used in this study and not previously reported. The mean OD is indicated by the line. Whiskers indicate the maximum and minimum values of replicates. (C) PGM binding ability of NoV VLPs determined by incubating GII.4 and GI.1-1968 VLPs with PGM-coated plates, followed by VLP detection with an anti-NoV antibody cocktail. (D) GII.4-2004 binds to PGM at higher VLP concentrations. Bars represent means with standard errors. Positive reactivity was defined as a mean optical density (OD) at 450 nm ≥3 time that of the background (dashed line).

Fig 3

Homotypic blockade of GII.4-2002 VLP-B trimer interaction by anti-GII.4-2002 MAbs. (A) GII.4-2002 VLPs were incubated with increasing concentrations of anti-GII.4-2002 MAbs, and the mean percent control binding was calculated by comparing the amount of VLP bound in the presence of antibody pretreatment to the amount of VLP bound in the absence of antibody pretreatment. Error bars represent the standard errors of the means. (B) Mean MAb concentration (μg/ml) needed to block 50% of GII.4-2002 ligand binding. The mean titer is indicated by the line in the graph. The upper and lower broken lines in the graph represent the maximum and minimum values. The asterisk indicates an antibody with a BT50 significantly different from that of GII.4-2002-G1.

Fig 4

Heterotypic blockade of GII.4 VLP-Bi-HBGA interactions by anti-GII.4-2002-G6. (A) Increasing concentrations of anti-GII.4-2002-G6 antibody were incubated with additional GII.4 VLPs positive for EIA reactivity and GI.1-1968 as a negative control. The mean percent control binding was calculated by comparing the amount of VLP bound to Bi-HBGA in the presence of antibody pretreatment to the amount of VLP bound in the absence of antibody pretreatment. Error bars represent the standard errors of the means. (B) The Mean MAb concentration (μg/ml) needed to block 50% of GII.4 VLP ligand binding is indicated by the line in the graph. The upper and lower broken lines in the graph represent the maximum and minimum values. Asterisks indicate VLPs with BT50s significantly different from that of GII.4-2002.

Fig 5

Blockade of GII.4 VLP-PGM interactions by GII.4-2002-G6. Increasing concentrations of anti-GII.4-2002-G6 were incubated with GII.4 VLPs positive for EIA reactivity, and the mean percent control binding was calculated by comparing the amount of VLP bound to PGM in the presence of antibody pretreatment to the amount of VLP bound in the absence of antibody pretreatment. Error bars represent the standard errors of the means. (B) The Mean MAb concentration (μg/ml) needed to block 50% of GII.4 VLP ligand binding is indicated by the line in the graph. The upper and lower broken lines in the graph represent the maximum and minimum values. Asterisks indicate VLPs with BT50s significantly different from that of GII.4-2002.

Fig 6

Detection of GII.4-2002 by Western blot analysis with anti-GII.4-2002 MAbs. GII.4-2002 VLPs were denatured, electrophoresed, and transferred to PVDF before being probed with 5 μg/ml and 1 μg/ml MAb. Mouse polyclonal anti-GII.4-2002 serum was used as a positive control for detection.

Fig 7

Epitope E was defined by variable residues 407, 412, and 413. (A) Variation in epitope E within GII.4 strains. (B) Epitope E (yellow) and HBGA binding sites (magenta) mapped onto the P domain dimer of GII.4-2002.

Fig 8

Characterization of epitope E, a GII.4-2002-specific potential neutralization epitope. (A) VLPs engineered to contain variations of epitope E were assembled to test the impact of the epitope and the nonepitope backbone on anti-GII.4-2002-G6 binding and neutralization. (B) Anti-GII.4-2002-G6 EIA reactivity to epitope E constructs. Asterisks indicate chimeric VLPs with reactivity significantly different from that of the parental VLPs. (C) Blockade of epitope E constructs binding to PGM by anti-GII.4-2002-G6. (D) The mean MAb concentration (μg/ml) needed to block 50% of GII.4 VLP ligand binding is indicated by the line in the graph. The upper and lower broken lines in the graph represent the maximum and minimum values. Asterisks indicate VLPs with BT50s significantly different from that of GII.4-2002.

Fig 9

Variation in epitope E alters capsid structure. Epitope E was defined by variable residues 407, 412, and 413 (406, 411, and 412 for GII.4.1987) and all residues within 8 Å of these sites, as these additional residues are likely to be impacted by the structural differences (driven by the variable sites) that contribute to MAb recognition differences. GII.4.1987 epitope E (A) differs from GII.4.2002 epitope E (B) at positions 406/407 and 355. The N-to-S difference at 406/407 likely alters the rotameric position of R411 in the GII.4.2002 VLP, allowing it to extend further from the surface. (C) GII.4.1987 (yellow) and GII.4.2002 (blue) superimposed. Because R411 is more buried in 1987, the MAb likely cannot interact as strongly with this residue in the GII.4.1987 VLP. GII.4-2002 (D) differs from GII.4-2006 (E) at five positions in the expanded epitope (positions 355 to 357, 412, and 413). (F) Superimposition suggests that R411 of GII.4.2006 (teal) is buried, and variation at 355 to 357 may alter key interactions involving H357 and D355. Red, differences between epitopes; yellow, key residues. (G) GII.4-2006/N412T (orange) superimposed upon GII.4.2006. Two potentially important residues are R411, which is more surface exposed in GII.4-2006/N412T, and T412, which is buried. (H) GII.4-2002 (blue) superimposed on GII.4-2006/N412T (orange). R411 is nearly identical, suggesting that N412T frees the R411 side chain to extend away from the surface, where it likely interacts with the MAb. Resides that regulate R411 make up site 1, and 87% of binding can be recovered with structural modifications to R411. A second site, site 2, is composed of residues 355 to 357, and particularly D355, which adds negative potential to the second site.