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. 2020;10(2):245-267.
doi: 10.1016/j.jcmgh.2020.03.006. Epub 2020 Apr 11.

Virus-Host Interactions Between Nonsecretors and Human Norovirus

Lisa C Lindesmith  1 , Paul D Brewer-Jensen  1 , Michael L Mallory  1 , Kara Jensen  1 , Boyd L Yount  1 , Veronica Costantini  2 , Matthew H Collins  3 , Caitlin E Edwards  1 , Timothy P Sheahan  1 , Jan Vinjé  2 , Ralph S Baric  4
Affiliations

Virus-Host Interactions Between Nonsecretors and Human Norovirus

Lisa C Lindesmith et al. Cell Mol Gastroenterol Hepatol. 2020.

Abstract

Background & aims: Human norovirus infection is the leading cause of acute gastroenteritis. Genetic polymorphisms, mediated by the FUT2 gene (secretor enzyme), define strain susceptibility. Secretors express a diverse set of fucosylated histoblood group antigen carbohydrates (HBGA) on mucosal cells; nonsecretors (FUT2-/-) express a limited array of HBGAs. Thus, nonsecretors have less diverse norovirus strain infections, including resistance to the epidemiologically dominant GII.4 strains. Because future human norovirus vaccines will comprise GII.4 antigen and because secretor phenotype impacts GII.4 infection and immunity, nonsecretors may mimic young children immunologically in response to GII.4 vaccination, providing a needed model to study cross-protection in the context of limited pre-exposure.

Methods: By using specimens collected from the first characterized nonsecretor cohort naturally infected with GII.2 human norovirus, we evaluated the breadth of serologic immunity by surrogate neutralization assays, and cellular activation and cytokine production by flow cytometry.

Results: GII.2 infection resulted in broad antibody and cellular immunity activation that persisted for at least 30 days for T cells, monocytes, and dendritic cells, and for 180 days for blocking antibody. Multiple cellular lineages expressing interferon-γ and tumor necrosis factor-α dominated the response. Both T-cell and B-cell responses were cross-reactive with other GII strains, but not GI strains. To promote entry mechanisms, inclusion of bile acids was essential for GII.2 binding to nonsecretor HBGAs.

Conclusions: These data support development of within-genogroup, cross-reactive antibody and T-cell immunity, key outcomes that may provide the foundation for eliciting broad immune responses after GII.4 vaccination in individuals with limited GII.4 immunity, including young children.

Keywords: Bile; Blockade Antibody; Cellular Immunity; Neutralizing Antibody; Receptor Binding.

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Figures

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Graphical abstract
Figure 1
Figure 1
Human norovirus GII.2 CH outbreak donor and strain characterization. (A) Donors are Lewis-positive, nonsecretors based on salivary secretion of Lewis a in the absence of H, Lewis b, A or B HBGA. (B) A GII.2 human norovirus strain was extracted from a stool sample collected 2 days after symptom onset and the GII.2 CH capsid gene was sequenced and expressed as VLPs. Original magnification: ×100,000. (C) GII.2 CH VLP-bound human type B saliva but not PGM, as reported for other GII.2 VLPs. (D) Donors had high ligand-binding blockade antibody titers to GII.2 CH at day 8 that persisted until the end of sample collection (1–6 mo), supporting GII.2 infection in all 4 donors. Markers denote the (A and C) means and SEM or (D) 95% CIs from a minimum of 2 replicates tested in 2 independent experiments. The dashed line is the lower limit of detection. IC50, 50% inhibitory concentration; Sec, secretor.
Figure 2
Figure 2
GII.2 infection broadly activates innate and adaptive immune responses. PBMCs collected from GII.2 CH–infected donors on days 8, 30, and 180 after infection and 7 blood bank donors were stained for activation markers and the percentage frequency of cells compared at each time point and against the basal response in the blood donors. Cellular subsets different from the donor responses are shown in the heat map (blue indicates an increase, orange indicates a decrease [P < .05], and white indicates no change [P ≥ .05]). One-way analysis of variance with the Kruskal–Wallis multiple comparison test. DC, dendritic cell.
Figure 3
Figure 3
Phenotypic frequencies of cell populations. Flow cytometry analysis first removed debris, multiplet events, and dead cells, before measuring lymphocyte and myeloid lineage cell frequencies. Using 3 distinct staining panels, the total T-cell (CD3, plus CD4+, CD8+, or CD4-CD8-), NK cell (CD3-CD16+), NK T-cell (CD3+CD16+), total B-cell (CD19+CD20+), monocyte (CD14+HLA-DR+), myeloid dendritic cell (CD11c+HLA-DR+CD14-), and plasmacytoid dendritic cell (CD123+CD14-) frequencies of total CD45+, live, singlet mononuclear events are shown. The CD4+ T, CD8+ T, and CD4-CD8- T-cell subsets are reported as a frequency of the total CD3+ T cells, and the TCRγδ+ T cells as a frequency of all CD3+CD4-CD8- cells. B cells first were classified using CD19 and CD20 coexpression (except plasmablasts) before further phenotyping as naïve (IgD+CD27-), transitional (CD24+CD38+), non–class-switched memory (IgD+CD27+), class-switched memory I (IgD-IgM+CD27+), class-switched memory II (IgD-IgM-CD27+), or plasmablasts (IgD-IgM-CD27+CD38+). Means with SD are shown. ∗P < .05 compared with healthy donor controls. Donor CH04 data are colored grey for comparison with the other donors. CH04 may be naïve for GII.2 infection, while the other donors likely are not naïve.
Figure 4
Figure 4
Activation marker expression patterns in the B-cell compartment. Unstimulated PBMCs were evaluated for markers of immune modulation in B cells. Cells first were classified as B-cell lineage using CD19 and CD20 coexpression, before further phenotyping as naïve (IgD+CD27-), transitional (CD24+CD38+), non–class-switched memory (IgD+CD27+), class-switched memory I (IgD-IgM+CD27+), or class-switched memory II (IgD-IgM-CD27+). Means with SD are shown. ∗P < .05 compared with healthy donor controls. Donor CH04 data are colored grey for comparison with the other donors. CH04 may be naïve for GII.2 infection, while the other donors likely are not naïve.
Figure 5
Figure 5
Activation marker expression patterns within myeloid and dendritic cell (DC) populations. Unstimulated PBMCs were characterized for expression of activation markers, costimulatory molecules, and cytokines after phenotyping monocytes (CD14+HLA-DR+), myeloid DCs (CD11c+HLA-DR+CD14-), and plasmacytoid DCs (CD123+CD14-). Means with SD are shown. ∗P < .05 compared with healthy donor controls. Donor CH04 data are colored grey for comparison with the other donors. CH04 may be naïve for GII.2 infection, while the other donors likely are not naïve. CCR, C-C chemokine receptor type 2.
Figure 6
Figure 6
Activation marker expression patterns in lymphoid cells. Unstimulated PBMCs were evaluated for markers of immune modulation in CD4+ T cells (CD3+CD4+CD8-), CD8+ T cells (CD3+CD4-CD8+), double-negative T cells (CD3+CD4-CD8-), NK cells (CD3-CD16+), and NK T cells (CD3+CD16+CD4-CD8-). Means with SD are shown. ∗P < .05 compared with healthy donor controls. Donor CH04 data are colored grey for comparison with the other donors. CH04 may be naïve for GII.2 infection, while the other donors likely are not naïve. KLRG, killer cell lectin-like receptor subfamily G member 1; PD1, programmed cell death 1.
Figure 7
Figure 7
GII.2 infection induces cross-GII antigen-specific cellular immune responses. PBMCs from GII.2-infected or control donors were stimulated with 5 μg/mL (A–C) GII.2 CH VLP, (D–F) GII.4 2012 Sydney VLP, or (G–I) GI.1 VLP incubated in the presence of Golgi transport inhibitors, and stained for CD4, CD8, and CD19 and secreted cytokines. Antigen-specific responses in T- and B-cell lymphocytes first were normalized to background frequencies in media control samples, and only the net change over background is reported. In these secretor-negative subjects, CD4+ and CD8+ T cells predominantly produced IFN-γ (purple), TNF-α (white), and IL4 (light blue) in response to GII.2 or GII.4 human norovirus VLP stimulation. CD19+ B cells primarily produced TNF-α (white) and IL17 (orange). One-way analysis of variance with the Kruskal–Wallis multiple comparison test. NT, not tested.
Figure 8
Figure 8
Cytokine production in response to ex vivo GII.2 VLP stimulation. PBMCs from GII.2-infected or control donors were stimulated with 5 μg/mL of GII.2 CH VLP, incubated in the presence of Golgi transport inhibitors, and stained for phenotypic cell markers CD3, CD4, CD8, and CD19, and secreted cytokines. Antigen-specific responses in T- and B-cell lymphocytes were first normalized to background frequencies in media control samples, and only the net change over background is reported. Means with SDs are shown. ∗P < .05 compared with 6 donor controls. Donor CH04 data are colored grey for comparison with the other donors. CH04 may be naïve for GII.2 infection, while the other donors likely are not naïve. The dashed line denotes no frequency detected.
Figure 9
Figure 9
Cytokine production in response to ex vivo GII.4 VLP stimulation. PBMCs from GII.2-infected or control donors were stimulated with 5 μg/mL of GII.4 2012 VLPs, incubated in the presence of Golgi transport inhibitors, and stained for phenotypic cell markers CD3, CD4, CD8, and CD19, and secreted cytokines. Antigen-specific responses in T- and B-cell lymphocytes were first normalized to background frequencies in media control samples, and only the net change over background is reported. Means with SD are shown. ∗P < .05 compared with 6 donor controls. The dashed line denotes no frequency detected.
Figure 10
Figure 10
PBMCs from control donors are more responsive to GII.4 2012 VLP ex vivo stimulation than GII.2 CH stimulation. PBMCs from control donors were stimulated with 5 μg/mL of GII.4 2012 or GII.2 CH VLP, incubated in the presence of Golgi transport inhibitors, and stained for phenotypic cell markers CD3, CD4, CD8, and CD19, and secreted cytokines. Antigen-specific responses in T- and B-cell lymphocytes were first normalized to background frequencies in media control samples, and only the net change over background is reported. The frequency of cytokine-producing cells after GII.4 2012 and GII.2 stimulation was compared. Means with SDs are shown. ∗P < .05 compared with GII.2 VLP response.
Figure 11
Figure 11
GII.2 infection induces cross-GII antibody responses in nonsecretors. (A) On day 8, ligand-binding blockade antibodies to GII.2 were greater than titers to other strains, supporting GII.2 infection in all donors. Blockade antibody titers included GII.17 and GII.4 2012, strains associated with infection of secretors. (B) Additional strain-specific GII.4 ligand-binding antibody was evaluated on day 8 in CH02 and CH03 and a control donor. (A and B) Markers denote the means and 95% CIs from a minimum of 2 replicates tested in 2 independent experiments. The dashed line is the lower limit of detection. (C) Between day 8 and day 180, titers to GII.2, GII.3, GII.14, and GII.17 decreased between 49% and 95%. In comparison, titer to GI.4 in CH02 was relatively stable at a 17% decrease (1.2-fold) change. IC50, 50% inhibitory concentration.
Figure 12
Figure 12
Bile salts enhance GII.2 CH VLP binding to nonsecretor ligands. (A) GII.2 CH and GII.4 2012 VLP binding to PGM in the absence (grey, light blue) or presence (black, dark blue) of increasing concentrations of crude bovine bile. Bile facilitates GII.2 binding to PGM. Inclusion of GCDCA or TCA purified bile salts had a modest impact on GII.2 CH and no impact on GII.4 2012 VLP binding to PGM. (B) GII.2 CH VLPs bind to infected donor salivary ligands in the presence of 1% bile. GI.1 VLP binding to secretor-negative saliva is not affected by bile. Markers denote the means and SDs of at least 1 representative of 2 independent experiments.
Figure 13
Figure 13
Bile salts do not support or enhance replication of GII.2 CH in HIE. Secretor-positive jejunal HIE monolayers (line J2) were pretreated 48 hours before inoculation with GCDCA/C2, pig bile, ox bile, or human bile. Monolayers were inoculated with GII.2 CH at 4.3 × 105 RNA copies/well and incubated for 72 hours. Jejunal HIE monolayers (line J2) also were inoculated with GII.4 Sydney[P16] at 2.1 × 105 RNA copies/well as a positive control for infection. Data represent the means with SDs of 2 experiments with 3 technical replicates for each experiment. hpi, hours postinfection; P16, Polymerase type 16.
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