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. 2012 Jan;86(2):1214-26.
doi: 10.1128/JVI.06189-11. Epub 2011 Nov 16.

Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity

Kari Debbink  1 , Eric F Donaldson, Lisa C Lindesmith, Ralph S Baric
Affiliations

Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity

Kari Debbink et al. J Virol. 2012 Jan.

Abstract

Noroviruses account for 96% of viral gastroenteritis cases worldwide, with GII.4 strains responsible >80% of norovirus outbreaks. Histo-blood group antigens (HBGAs) are norovirus binding ligands, and antigenic and preferential HBGA binding profiles vary over time as new GII.4 strains emerge. The capsid P2 subdomain facilitates HBGA binding, contains neutralizing antibody epitopes, and likely evolves in response to herd immunity. To identify amino acids regulating HBGA binding and antigenic differences over time, we created chimeric virus-like particles (VLPs) between the GII.4-1987 and GII.4-2006 strains by exchanging amino acids in putative epitopes and characterized their antigenic and HBGA binding profiles using anti-GII.4-1987 and -2006 mouse monoclonal antibodies (MAbs) and polyclonal sera, 1988 outbreak human sera, and synthetic HBGAs. The exchange of amino acids 393 to 395 between GII.4-1987 and GII.4-2006 resulted in altered synthetic HBGA binding compared to parental strains. Introduction of GII.4-1987 residues 294, 297 to 298, 368, and 372 (epitope A) into GII.4-2006 resulted in reactivity with three anti-GII.4-1987 MAbs and reduced reactivity with four anti-GII.4-2006 MAbs. The three anti-GII.4-1987 MAbs also blocked chimeric VLP-HBGA interaction, while an anti-GII.4-2006 blocking antibody did not, indicating that epitope A amino acids comprise a potential neutralizing epitope for GII.4-1987 and GII.4-2006. We also tested GII.4-1987-immunized mouse polyclonal sera and 1988 outbreak human sera for the ability to block chimeric VLP-HBGA interaction and found that epitope A amino acids contribute significantly to the GII.4-1987 blockade response. Our data provide insights that help explain the emergence of new GII.4 epidemic strains over time, may aid development of norovirus therapeutics, and may help predict the emergence of future epidemic strains.

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Figures

Fig 1
Fig 1
Putative epitopes A and D. (A) Amino acid differences between GII.4-1987 Camberwell and GII.4-2006 Minerva in the P2 region of VP1. Green shading indicates amino acids within putative epitope A, and orange indicates amino acids within putative epitope D. Shared residue 296S was included because it falls within putative epitope A. (B) Epitope A is shown in green, consisting of 6 amino acids at positions 294, 296, 297, 298, 368, and 372. Epitope D is shown in orange, consisting of 3 amino acids at positions 393 to 395. Black shading indicates the HBGA binding pocket. (C) Epitope A chimeric VLPs. The parental GII.4-2006 and GII.4-1987 amino acid residues are shown in purple and yellow, respectively. GII.4-2006 Minerva residues (purple) were replaced in each chimera by GII.4-1987 Camberwell residues at the positions shown in yellow. A total of four epitope A chimeric VLPs were created. (D) Representative EM images of parental and chimeric VLPs. (a) GII.4-1987; (b) GII.4-2006; (c) GII.4-1987(+06D); (d) GII.4-2006(+87D); (e) GII.4-2006(+87A.1); (f) GII.4-2006(+87A.2); (g) GII.4-2006(+87A.3); (h) GII.4-2006(+87A.4).
Fig 2
Fig 2
Epitope D chimeric VLP reactivity with synthetic HBGAs and anti-GII MAbs. (A) HBGA reactivity. Synthetic CHO was diluted to 10 μg/ml and assayed by the CHO binding assay for interaction with GII.4-1987, GII.4-2006, GII.4-1987(+06D), and GII.4-2006(+87D). Amino acid residues from GII.4-1987 were exchanged with GII.4-2006 residues at positions 393 to 395. The dashed line represents 3 times the background level of binding and indicates a positive signal. Asterisks indicate significant differences. (B) Anti-GII.4 MAb reactivity. Anti-GII.4-1987 and -2006 MAbs were diluted to 2 μg/ml and assayed by EIA for reactivity with GII.4-1987, GII.4-2006, GII.4-1987(+06D), and GII.4-2006(+87D) VLPs. Amino acid residues from GII.4-1987 were exchanged with GII.4-2006 at positions 393 to 395. Bars represent the mean optical density values with standard errors of the means. The dashed line represents 3 times the background level of binding and indicates a positive signal.
Fig 3
Fig 3
GII.4-2006(+87A.1) reactivity with anti-GII.4 MAbs. Anti-GII.4-1987 and -2006 IgG was diluted to 2 μg/ml and assayed by EIA for reactivity with GII.4-1987, GII.4-2006, and GII.4-2006(+87A.1) VLPs. Amino acid residues from GII.4-1987 were inserted into the GII.4-2006 backbone at positions 294, 297, 298, 368, and 372 to create GII.4-2006(+87A.1). Bars represent the mean optical density values with standard errors of the means. The dashed line represents 3 times the background level of binding and indicates a positive signal. Asterisks indicate significant differences.
Fig 4
Fig 4
Epitope A chimeric VLP reactivity with synthetic HBGAs. Synthetic CHOs were diluted to 10 μg/ml and assayed by the CHO binding assay for interaction with GII.4-1987, GII.4-2006, and GII.4-2006(+87A.1). Amino acid residues from GII.4-1987 were inserted into the GII.4-2006 backbone at positions 294, 297 to 298, 368, and 372. The dashed line represents 3 times the background level of binding and indicates a positive signal.
Fig 5
Fig 5
GII.4-2006(+87A.2) reactivity with anti-GII.4 MAbs. Anti-GII.4-1987 and -2006 IgG was diluted to 2 μg/ml and assayed by EIA for reactivity with GII.4-1987, GII.4-2006, and GII.4-2006(+87A.2) VLPs. Amino acid residues from GII.4-1987 were inserted into the GII.4-2006 backbone at positions 297 and 298 to create GII.4-2006(+87A.2). Bars represent the mean optical density values with standard errors of the means. The dashed line represents 3 times the background level of binding and indicates a positive signal. Asterisks indicate significant differences.
Fig 6
Fig 6
GII.4-2006(+87A.3) reactivity with anti-GII.4 MAbs. Anti-GII.4-1987 and -2006 IgG was diluted to 2 μg/ml and assayed by EIA for reactivity with GII.4-1987, GII.4-2006, and GII.4-2006(+87A.3) VLPs. Amino acid residues from GII.4-1987 were inserted into the GII.4-2006 backbone at positions 368 and 372 to create GII.4-2006(+87A.3). Bars represent the mean optical density values with standard errors of the means. The dashed line represents 3 times the background level of binding and indicates a positive signal.
Fig 7
Fig 7
GII.4-2006(+87A.4) reactivity with anti-GII.4 MAbs. Anti-GII.4-1987 and -2006 IgG was diluted to 2 μg/ml and assayed by EIA for reactivity with GII.4-1987, GII.4-2006, and GII.4-2006(+87A.4) VLPs. An amino acid residue from GII.4-1987 was inserted into the GII.4-2006 backbone at position 294 to create GII.4-2006(+87A.4). Bars represent the mean optical density values with standard errors of the means. The dashed line represents 3 times the background level of binding and indicates a positive signal.
Fig 8
Fig 8
Anti-GII.4 MAb blockade of VLP/synthetic HBGA interactions. (A, B) The ability of anti-GII.4-1987-G1 (A) and anti-GII.4-2006-G7 (B) to block VLP interaction with synthetic HBGA was measured by surrogate neutralization assay and expressed as a percentage of the control binding (100%). An antibody is considered a “blocking antibody” if it is able to block at least 50% of the control (dashed line). Anything below the dashed line represents potential neutralization. Error bars represent standard errors of the means from experiments run in triplicate.
Fig 9
Fig 9
GII.4-1987 mouse polyclonal serum blockade. (A) GII.4-1987 polyclonal mouse sera was diluted 1:1,000 and assayed by EIA for reactivity with GII.4-1987, GII.4-2006, and GII.4-2006(+87A.1) VLPs. Bars represent the mean optical density values with standard errors of the means. The dashed line represents 3 times the background level of binding and indicates a positive signal. (B) GII.4-1987 polyclonal mouse serum blockade of GII.4 VLP/synthetic HBGA interactions. Ability of GII.4-1987 polyclonal mouse sera to block VLP/synthetic CHO was measured by surrogate neutralization assay and expressed as a percentage of the control binding (100%). An antibody is considered a “blocking antibody” if it is able to block at least 50% of the control (dashed line). Anything below the dashed line represents potential neutralization. Error bars represent standard errors of the means from experiments run in triplicate. (C) GII.4-1987 polyclonal mouse serum BT50 values for GII.4-1987, GII.4-2006, and GII.4-2006(+87A.1). Blocking titers of GII.4-1987 mouse polyclonal sera were determined as the serum concentration at which 50% of the VLP/synthetic HBGA was blocked. Box and whiskers plots represent the mean serum concentration at which BT50 was achieved for each VLP.
Fig 10
Fig 10
1988 human outbreak serum blockade. (A) 1988 human outbreak sera was diluted 1:500 and assayed by EIA for reactivity with GII.4-1987, GII.4-2006, and GII.4-2006(+87A.1) VLPs. Bars represent the mean optical density values with standard errors of the means. The dashed line represents 3 times the background level of binding and indicates a positive signal. (B) 1988 human outbreak serum blockade of GII.4 VLP/synthetic HBGA interactions. The ability of human sera to block VLP/synthetic CHO was measured by surrogate neutralization assay and expressed as a percentage of the control binding (100%). An antibody is considered a “blocking antibody” if it is able to block at least 50% of the control (dashed line). Anything below the dashed line represents potential neutralization. Error bars represent standard errors of the means from experiments run in triplicate. (C) 1988 human outbreak serum BT50 values for GII.4-1987, GII.4-2006, and GII.4-2006(+87A.1). Blocking titers of human sera were determined as the serum concentration at which 50% of the VLP/synthetic HBGA was blocked. Box and whiskers plots represent the mean serum concentration at which BT50 was achieved for each VLP.
Fig 11
Fig 11
Homology models comparing GII.4-1987, GII.4-2006, and GII.4-1987(+06D). (A) Overlay of binding pocket of GII.4-1987 (yellow), GII.4-2006 (purple), and GII.4-1987(+06D) (gray). Side chains are shown for residues in sites 1 and 2 (residues 342 to 347, 374, 390 to 395, and 440 to 444). Residues 393 to 395 are shown in teal [GII.4-1987(+06D)], orange (GII.4-1987), and red (GII.4-2006). (B) Residue 390 (yellow) forms a hydrogen bond with residue 395 (yellow) in GII.4-1987(+06D) that is not found in GII.4-2006. GII.4-2006 residues 393 and 395 form hydrogen bonds not found in GII.4-1987(+06D). (C) Residue 343 (yellow) forms a hydrogen bond (red arrow) with the FUC of HBGA B (orange) in GII.4-1987(+06D) but not with that of GII.4-2006. (D) Three hydrogen bonds are formed between residue 345 (yellow) and the FUC of HBGA B in GII.4-2006, but only one is formed with GII.4-1987(+06D). (E) Residues 343 and 444 (both yellow) share a hydrogen bond in GII.4-1987(+06D) but not in GII.4-2006. (F) Residue 442 (yellow) participates in hydrogen bonding with the FUC of HBGA B in GII.4-1987(+06D) but not with that of GII.4-2006. In GII.4-1987(+06D), residue 442 (yellow) also forms a hydrogen bond with residue 443, which is not found in GII.4-2006.
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