Heterotypic humoral and cellular immune responses following Norwalk virus infection - PubMed (original) (raw)

Heterotypic humoral and cellular immune responses following Norwalk virus infection

Lisa C Lindesmith et al. J Virol. 2010 Feb.

Abstract

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-gamma) was measured. As seen with blockade responses, IFN-gamma secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.

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Figures

FIG. 1.

FIG. 1.

Bayesian analysis of the GI genogroup. The 48 sequences of the GI genogroup were aligned and analyzed by Bayesian inference, which demonstrated the evolutionary relationship between the eight genotypes. Only the reference sequences are shown, along with those sequences used to generate VLPs, indicated in bold with an asterisk. Black boxes indicate genotypes. Scale bar, 0.1 substitutions per site.

FIG. 2.

FIG. 2.

Variation in the P2 subdomain of the first four GI genotypes. Variation within the P2 subdomain of eight GI.1 to GI.4 representative sequences is shown with the genotype-specific changes colored as follows: GI.1 Norwalk, red; GI.2 Southampton, blue; GI.3 Desert Shield, green; and the GI.4 Chiba, purple. Amino acids that occur within multiple genotypes on one occurrence (yellow) and multiple occurrences (orange) are shown. Unique differences that occur within a genotype are indicated by a lighter shade of the primary genotype color. Position numbers filled in gray indicate residues that interact with HBGAs in the GI.1 Norwalk capsid, and columns that are shown in gray indicate residues that are strictly conserved within these four genotypes. Alignment indicates numbering based upon the multiple alignment, and Norwalk indicates numbering based upon the GI.1 Norwalk sequence. Representative sequences were selected to demonstrate the sequence heterogeneity within the genotype.

FIG. 3.

FIG. 3.

Structural comparison between the Norwalk P domain structure and GI genogroup models. Homology models were generated for GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000 P domain sequences using chain A and chain B of the Norwalk P domain dimer structure, and these were compared. In all cases, the residues reported to interact with the HBGAs were conserved (magenta). Chain A of each model is shown in the brighter shade on the left, and chain B is shown as the lighter shade on the right. Magenta indicates the binding pocket. (A) Norwalk crystal structure with the H type 1 pentasaccharide shown engaging with the receptor binding pocket. (B) The GI.2-1999 Southampton model shows that inserts have added loops to the top of the structure, which extends from the surface adjacent to the conserved binding pocket. (C) The GI.3-1999 Desert Shield model is structurally similar to that of the Norwalk structure, with a conserved receptor-binding pocket. (D) The GI.4-2000 Chiba model is also structurally similar to the Norwalk structure, with a conserved receptor-binding region.

FIG. 4.

FIG. 4.

Structural differences between the Norwalk P domain structure and the GI genogroup models. Analysis of the homology models in comparison to the Norwalk P domain dimer structure demonstrated that much of the variation in the genogroup occurs in loops that extend to the surface. Red indicates a conserved structural domain, purple indicates the binding pocket, and yellow indicates variation within the genotype. (A) The variation over 30 years within the GI.1 genotype resulted in only minor structural differences. The only observed change was a change of Asp to Glu at Norwalk capsid position 350 (arrow). (B) The GI.2-1999 Southampton genotype mostly differs from GI.1 by the addition of several residues that occur in loops shown in green. (C) The GI.3-1999 Desert Shield genotype mostly differs from GI.1 by the addition of residues in loops shown in brown. (D) The GI.4-2000 Chiba genotype mostly differs from GI.1 by the addition of only a few residues that occur in loops shown in orange.

FIG. 5.

FIG. 5.

GI VLP-salivary binding patterns at room temperature. GI VLPs were assayed for ability to bind to saliva samples phenotyped for secretor status (Se) and ABO blood type by EIA. OD, optical density.

FIG. 6.

FIG. 6.

GI VLP-carbohydrate binding patterns at room temperature. VLPs were assayed for the ability to bind to synthetic biotinylated HBGA bound to avidin-coated plates. The mean optical density (OD) is indicated by the line in the box. The upper and lower boundaries of the box represent the maximum and minimum values. (A) VLP binding to core chains including an α-1,2-fucose. (B) VLP binding to either core chains or H antigens modified with the Lewis antigen. (C) VLP binding to A or B antigen trimer.

FIG. 7.

FIG. 7.

GI VLPs share common antibody epitopes. The median geometric mean titer of anti-VLP IgG (μg/ml) in prechallenge (day 0; dotted bars) and convalescent (day 14; shaded bars) serum samples collected from volunteers infected with GI.1-1968 and the number of subjects who seroconverted to each VLP were determined. The median titer is indicated by the line in the box. The upper and lower boundaries of the box represent the maximum and minimum values. The asterisk indicates a significant increase (P < 0.01, M-W test) in titer between day 0 and day 14 samples.

FIG. 8.

FIG. 8.

Cross-blockade of GI VLPs binding to HBGA by GI.1-1968 convalescent-phase serum. Serum samples collected from GI.1-1968-infected volunteers were assayed for blockade of H type 3 interaction with GI.1-1968, GI.1-2001, and GI.2-1999 and Lea interaction with GI.3-1999 and GI.4-2000, and the median percent control binding was calculated relative to the no-serum control binding. (A) Prechallenge serum. (B) Day 14 convalescent serum. (C) Scatter plot of the median percent serum needed for BT50 and the number of volunteers with a BT50 for each VLP (indicated below the graph). The median titer is indicated by the line in the box. Error bars represent the range.

FIG. 9.

FIG. 9.

GI VLP blockade responses vary by individual. Serum samples collected from GI.1-1968-infected volunteers at day 0 (prechallenge; left-hand column) and day 14 (right-hand column) were assayed for blockade of H type 3 interaction with GI.1-1968, GI.1-2001, and GI.2-1999 and Lea interaction with GI.3-1999 and GI.4-2000, and the percent control binding was calculated relative to the no-serum control binding.

FIG. 9.

FIG. 9.

GI VLP blockade responses vary by individual. Serum samples collected from GI.1-1968-infected volunteers at day 0 (prechallenge; left-hand column) and day 14 (right-hand column) were assayed for blockade of H type 3 interaction with GI.1-1968, GI.1-2001, and GI.2-1999 and Lea interaction with GI.3-1999 and GI.4-2000, and the percent control binding was calculated relative to the no-serum control binding.

FIG. 10.

FIG. 10.

GI.1-1968 convalescent-phase serum does not block cross-genogroup GII.4-1997 VLP binding to HBGA. (A) Convalescent-phase serum samples collected from individuals infected with GI.1-1968 (circles), GII.2-1976 (squares), and GII.4 (triangles) noroviruses (n = 6 each strain) were assayed for blockade of GII.4-1997-H type 3 interaction, and the median percent control binding was calculated relative to the no-serum control binding. Error bars represent the range. (B) Scatter plot of the median percent serum needed for BT50 for blockade of GII.4-1997-H type 3 interaction. The median titer is indicated by the line in the box. Error bars represent the range. Serum BT50 responses significantly different (P < 0.05, KW test) from GI.1-1968 blockade are marked with an asterisk.

FIG. 11.

FIG. 11.

GI.1-1968 infection induces GI-reactive PBMCs collected from volunteers infected with GI.1-1968 were stimulated individually with a panel of GI VLPs and IFN-γ secretion measured by EIA on days 0, 4, 14, and 35 postchallenge. Only VLPs with two consecutive time points with elevated IFN-γ secretion levels are shown for each volunteer.

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