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. 2016 Oct 3:7:1570.
doi: 10.3389/fmicb.2016.01570. eCollection 2016.

Norovirus-Specific Memory T Cell Responses in Adult Human Donors

Maria Malm  1 , Kirsi Tamminen  1 , Timo Vesikari  1 , Vesna Blazevic  1
Affiliations

Norovirus-Specific Memory T Cell Responses in Adult Human Donors

Maria Malm et al. Front Microbiol. .

Abstract

Norovirus (NoV) is a leading cause of acute gastroenteritis in people of all ages worldwide. NoV-specific serum antibodies which block the binding of NoV virus-like particles (VLPs) to the cell receptors have been thoroughly investigated. In contrast, only a few publications are available on the NoV capsid VP1 protein-specific T cell responses in humans naturally infected with the virus. Freshly isolated peripheral blood mononuclear cells of eight healthy adult human donors previously exposed to NoV were stimulated with purified VLPs derived from NoV GII.4-1999, GII.4-2012 (Sydney), and GI.3, and IFN-γ production was measured by an ELISPOT assay. In addition, 76 overlapping synthetic peptides spanning the entire 539-amino acid sequence of GII.4 VP1 were pooled into two-dimensional matrices and used to identify putative T cell epitopes. Seven of the eight subjects produced IFN-γ in response to the peptides and five subjects produced IFN-γ in response to the VLPs of the same origin. In general, stronger T cell responses were induced with the peptides in each donor compared to the VLPs. A CD8+ T cell epitope in the shell domain of the VP1 (134SPSQVTMFPHIIVDVRQL151) was identified in two subjects, both having human leukocyte antigen (HLA)-A∗02:01 allele. To our knowledge, this is the first report using synthetic peptides to study NoV-specific T cell responses in human subjects and identify T cell epitopes.

Keywords: ELISPOT IFN-gamma; PBMC; T cell epitope; VLP; cellular immunity; norovirus; peptide pools.

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Figures

FIGURE 1
FIGURE 1
Electron microscopy image of baculovirus-insect cell system-produced and sucrose gradient-purified norovirus GII.4-99 (A), GII.4 Sydney (B), and GI.3 (C) virus-like particles (VLPs). VLPs were negatively stained with 3% uranyl acetate (pH 4.5) and the preparations were examined using an FEI Tecnai F12 electron microscope at a magnification of 23,000×.
FIGURE 2
FIGURE 2
The gating strategy for flow cytometry analysis from representative data. PBMCs from a donor were stimulated with SEB for 16 h in the presence of brefeldin A and then stained for IFN-γ and lineage markers CD3 and CD8. A forward and side scatter was used for gating the lymphocyte population. CD3+ lymphocyte population was further gated to CD8+ and CD8- populations and IFN-γ staining was analyzed for both populations as previously described by others (Meddows-Taylor et al., 2007). Dot plots show gated populations and events as percentages of the parent population.
FIGURE 3
FIGURE 3
Norovirus genotype-specific immunoglobulin G (IgG) antibody titers. The plasma of eight donors was used to determine individual end-point titer (A) and geometric mean titers (GMTs) with 95% confidence intervals (B) against NoV GII.4-1999, GII.4 Sydney, and GI.3 VLPs in an enzyme-linked immunosorbent assay (ELISA). Statistical significance was determined by Fisher’s exact test, and a p-value ≤ 0.05 was considered to be statistically significant (∗).
FIGURE 4
FIGURE 4
Correlation of GII.4-1999-specific antibody and T cell responses. Spearman’s rank correlation (r) was determined between the norovirus GII.4-1999-specific antibody titer and ELISPOT IFN-γ spot-forming cell (SFC)/106 peripheral blood mononuclear cells (PMBCs) in response to GII.4-99 VLPs (A) and a complete capsid peptide pool (B) stimulation.
FIGURE 5
FIGURE 5
Enzyme-linked immunosorbent spot interferon gamma (IFN-γ) responses to GII.4-1999 matrix peptide pool stimulation. Peripheral blood mononuclear cells (PBMCs) of eight donors (#1-8) were stimulated with 76 18-mer GII.4-99 capsid-derived single peptides organized into 18 matrix pools (M1–M18) or left unstimulated (CM, culture media). Shown are the mean spot-forming cells (SFC)/106 PBMCs of two replicate wells with the standard errors of the mean. The cut-off line (dotted line) represents the positive SFC/106 value of >50 SFC/106 cells.
FIGURE 6
FIGURE 6
Enzyme-linked immunosorbent spot IFN-γ responses toward NoV GII.4-1999 T cell epitope 99-20. Fresh peripheral blood mononuclear cells (PBMCs) of donors #2 (A) and #4 (B) were stimulated with an increasing concentration (0–4 μg/ml) of peptide 99-20 containing the putative NoV-specific T cell epitope. Shown are the spot-forming cells (SFC)/106 PBMCs of two replicate wells with standard errors of the mean. The cut-off line (dotted line) represents the positive SFC/106 value of >50 SFC/106 cells.
FIGURE 7
FIGURE 7
Expression of IFN-γ, IL-2, and TNF-α by intracellular staining after stimulation with NoV GII.4-1999 peptide epitope 99-20. Peripheral blood mononuclear cells (PBMCs) of donors #2 having A∗01:01/∗02:01; B∗08:01/∗56:01; C∗01:02/∗07:01 HLA class I phenotype alleles (A) and #4 having A∗02:01/∗32:01; B∗35:01/∗40:02; C∗03:04/∗04:01 HLA class I phenotype alleles (B) were stimulated with 2 μg/ml of 99-20 single peptide for 16 h and the cytokine response was measured by flow cytometry. PBMCs in culture media only (CM) (left panel) and peptide-stimulated PBMCs (right panel) were gated to CD3+CD8+ T cells and the percentages of IFN-γ, IL-2, or TNF-α cytokine-expressing cells are indicated.
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