Intestinal Norovirus Binding Patterns in Nonsecretor Individuals

Abstract

Human norovirus (HuNoV) infection is associated with an active FUT2 gene, which characterizes the secretor phenotype. However, nonsecretor individuals are also affected by HuNoV infection although in a lesser proportion. Here, we studied GII.3, GII.4, and GII.17 HuNoV interactions in nonsecretor individuals using virus-like particles (VLPs). Only GII.4 HuNoV specifically interacted with nonsecretor saliva. Competition experiments using histo-blood group antigen (HBGA)-specific monoclonal antibodies (MAbs) demonstrate that GII.4 VLPs recognized the Lewis a (Le^a) antigen. We also analyzed HuNoV VLP interactions on duodenum tissue blocks from healthy nonsecretor individuals. VLP binding was observed for the three HuNoV genotypes in 10 of the 13 individuals, and competition experiments demonstrated that VLP recognition was driven by an interaction with the Le^a antigen. In 3 individuals, binding was restricted to either GII.4 alone or GII.3 and GII.17. Finally, we performed a VLP binding assay on proximal and distal colon tissue blocks from a nonsecretor patient with Crohn's disease. VLP binding to inflammatory tissues was genotype specific since GII.4 and GII.17 VLPs were able to interact with regenerative mucosa, whereas GII.3 VLP was not. The binding of GII.4 and GII.17 HuNoV VLPs was linked to Le^a in regenerative mucosae from the proximal and distal colon. Overall, our data clearly showed that Le^a has a pivotal role in the recognition of HuNoV in nonsecretors. We also showed that Le^a is expressed in inflammatory/regenerative tissues and interacts with HuNoV in a nonsecretor individual. The physiological and immunological consequences of such interactions in nonsecretors have yet to be elucidated. IMPORTANCE Human norovirus (HuNoV) is the main etiological agent of viral gastroenteritis in all age classes. HuNoV infection affects mainly secretor individuals where ABO(H) and Lewis histo-blood group antigens (HBGAs) are present in the small intestine. Nonsecretor individuals, who only express Lewis (Le) antigens, are less susceptible to HuNoV infection. Here, we studied the interaction of common HuNoV genotypes (GII.3, GII.4, and GII.17) in nonsecretor individuals using synthetic viral particles. Saliva binding assays showed that only GII.4 interacted with nonsecretor saliva via the Lewis a (Le^a) antigen Surprisingly, the three genotypes interacted with nonsecretor enterocytes via the Le^a antigen on duodenal tissue blocks, which were more relevant for HuNoV/HBGA studies. The Le^a antigen also played a pivotal role in the recognition of GII.4 and GII.17 particles by inflammatory colon tissue from a nonsecretor Crohn's disease patient. The implications of HuNoV binding in nonsecretors remain to be elucidated in physiological and pathological conditions encountered in other intestinal diseases.

Keywords: Crohn’s disease; FUT2; HBGA; colon; duodenum; healthy; nonsecretor; norovirus.

FIG 1

Saliva binding assays on nonsecretor saliva. The binding of cesium chloride-purified VLP was measured by ELISA in duplicate for each saliva sample, and the mean values are shown on the graph. The VLP genotype is indicated on the top right corner of each histogram. The number of the sample is indicated on the horizontal axis, and OD₄₅₀ values are indicated on the vertical axis of each histogram.

FIG 2

Competition experiments using GII.4 VLP in saliva binding assays. Each experiment was conducted in triplicate for each saliva sample, and the mean values are shown on the graph. Vertical bars represent standard deviation. The saliva sample is indicated above each graph. On each graph, antibodies are color coded (anti-Le^a [blue], anti-Le^b [red], and anti-Le^x [green]), and the legend is on the right side of the graphs. The OD₄₅₀ values and MAb quantity used for the experiments are indicated on the vertical axis and the horizontal axis, respectively.

FIG 3

GII.4, GII.3, and GII.17 binding in healthy nonsecretor duodenal samples. Incubated VLP are indicated on the left of each line. Duodenal samples 1NS to 13NS are indicated on the top of each row. Positive VLP binding is characterized by brown staining. The panels of this figure and the following figure are shown at magnification ×200 unless indicated otherwise.

FIG 4

Lewis antigen detection in healthy nonsecretor duodenal samples. The detected Lewis antigens are indicated to the left of each line. Duodenal samples 1NS to 13NS are indicated on top of each row. Positive HBGA detection is shown by brown staining.

FIG 5

VLP competition experiments using healthy nonsecretor duodenal samples from patients 3NS, 12NS, and 11NS. Preincubation of MAbs and control are indicated on the top of each row. The genotype of the VLP used for the assay is indicated to the left of each line. The sample used for the experiments is indicated in parentheses. Positive VLP detection is indicated by brown staining and with arrows.

FIG 6

VLP detection in quiescent samples of the ileum, proximal colon, distal colon, and appendix from nonsecretor Crohn’s disease patient 14NS. The anatomical site of the sample is indicated on top of each row. Incubated VLPs are indicated at the left of each line. Positive VLP detection is shown by brown staining and with arrows.

FIG 7

Blood group B, Lewis antigen, and sialylated Lewis antigen detection in quiescent samples of the ileum, proximal colon, distal colon, and appendix from nonsecretor Crohn’s disease patient 14NS. The anatomical origin of each tissue sample is indicated to the left of each line. The detected HBGAs are indicated on the top of each row. Positive HBGA detection is shown by brown staining.

FIG 8

VLP detection in regenerative mucosa of samples of proximal and distal colon from nonsecretor Crohn’s disease patient 14NS (magnification, ×400). The anatomical sites of each sample are indicated on the left. The VLP genotype is indicated on the top (row). Positive VLP detection is indicated by brown staining and pointed by arrows.

FIG 9

Blood group B, Lewis antigen, and sialylated Lewis antigen detection in regenerative mucosa of samples of proximal colon and distal colon from nonsecretor Crohn’s disease patient 14NS. The anatomical site of each sample is indicated on the left. The detected HBGAs are indicated on the top of each row. Positive HBGA detection is shown by light- or dark-brown staining and with arrows. The panels are shown at magnification ×400.

FIG 10

VLP competition experiments performed on regenerative mucosa in samples of proximal colon and distal colon from nonsecretor Crohn’s disease patient 14NS (magnification, ×400). Preincubation of MAbs and mock control are indicated on top of each row. The following incubation of VLP is indicated at the left. Positive VLP detection is indicated by brown staining and with arrows.