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. 2018 Nov 27:9:2782.
doi: 10.3389/fimmu.2018.02782. eCollection 2018.

Identification of a First Human Norovirus CD8+ T Cell Epitope Restricted to HLA-A*0201 Allele

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

Identification of a First Human Norovirus CD8+ T Cell Epitope Restricted to HLA-A*0201 Allele

Maria Malm et al. Front Immunol. .

Abstract

Norovirus (NoV) causes a substantial global burden of acute gastroenteritis in all age groups and the development of NoV vaccine is a high priority. There are still gaps in understanding of protective NoV-specific immunity. Antibody mediated immune responses have been widely studied, but in contrast, the research on NoV-specific human T cell-mediated immunity is very limited. We have recently reported NoV capsid VP1-specific 18-mer peptide (134SPSQVTMFPHIIVDVRQL151) to induce strong CD8+ T cell immune responses in healthy adult donors. This work extends to identify the precise NoV T cell epitope and the restricting human leucocyte antigen (HLA). Pentamer technology was used to detect HLA-A*0201-restricted T cell-mediated responses to 10-mer peptide 139TMFPHIIVDV148 of four healthy adult blood donors. Immunogenicity of the 10-mer epitope was confirmed by ELISPOT IFN-γ and intracellular cytokine staining (ICS) on flow cytometry. A population of CD3+CD8+ T lymphocytes binding to HLA-A*0201/TMFPHIIVDV pentamers was identified in two HLA-A*0201-positive donors. Recognition of the 10-mer epitope by T cells resulted in a strong IFN-γ secretion as shown by ELISPOT assay. In addition, ICS confirmed that high proportion (31 and 59%) of the TMFPHIIVDV epitope-responsive CD3+CD8+ T cells in the two donors had multifunctional phenotype, simultaneously producing IFN-γ, IL-2 and TNF-α cytokines. In the present study novel human NoV HLA-A*0201-restricted minimal 10-mer epitope 139TMFPHIIVDV148 in the capsid VP1 was identified. The HLA-peptide pentamer staining of T cells from healthy donor PBMCs and cytokine responses in ex-vivo ELISPOT and ICS assays suggest that this epitope is recognized during NoV infection and activates memory phenotype of the epitope-specific multifunctional CD8+ T cells. The importance of this epitope in protection from NoV infection remains to be determined.

Keywords: CD8 T cell epitope; ELISPOT IFN-gamma; HLA-A2*0201; cellular immunity; multifunctional T cells; norovirus; pentamer.

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Figures

Figure 1
Figure 1
Phylogenetic analysis of norovirus major capsid protein VP1 amino acid sequences. The tree was inferred by Neighbor-Joining method with a bootstrap of 500 by using MegaX. The scale bar shows the genetic distance, expressed as amino acid substitutions per site.
Figure 2
Figure 2
Pentamer staining of norovirus-derived 139TMFPHIIVDV148 epitope-specific CD8+ T cells. Peripheral blood mononuclear cells (PBMCs) from two HLA-A*0201-positive healthy adult donors and the negative control were directly stained using 0.25 and 0.5 μg HLA-A*0201/TMFPHIIVDV pentamer or negative control pentamer. The dot plots show the percentage of pentamer-positive T cells for two HLA-A*0201-positive donors and a representative negative control donor after gating on live CD3+CD8+ T lymphocyte population.
Figure 3
Figure 3
Norovirus epitope 139TMFPHIIVDV148 –specific enzyme-linked immunosorbent spot (ELISPOT) interferon-gamma (IFN-γ) responses. PBMCs of Donor 1 (A,B) and Donor 2 (A,C) positive for HLA-A*0201 allele were stimulated with an increasing concentration (0–4 μg/ml) of the 10-mer epitope peptide, 4 μg/ml irrelevant 9-mer peptide (Ctrl), or left unstimulated (CM, culture media). Images of the ELISPOT replicate wells with the stained spot-forming cells (SFC) are shown (A). Mean SFC/106 PBMCs of two replicate wells with the standard errors of the mean for Donor 1 (B) and Donor 2 (C). The cut-off line (dotted line) indicates the positive SFC/106 value of ≥50 SFC/106 cells and twice above the background control (CM only wells).
Figure 4
Figure 4
Functional characterization of norovirus 10-mer epitope 139TMFPHIIVDV148 –specific CD8+ T cells by intracellular cytokine staining (ICS). Production of IL-2, IFN-γ, and TNF-α measured by ICS after 16 h stimulation of the PBMC with the peptide 139TMFPHIIVDV148 (4 μg/ml). Shown are the results of the two HLA-A*0201-positive donors, Donor 1 (A) and Donor 2 (B). Live epitope-specific CD3+ CD8+ T cells were segregated into IFN-γ+ and IFN-γ− T cells. Seven distinct populations, based on production of the cytokines in any combination are depicted on the pie charts and the accompanying tables. Fractions of the cells producing one, two, or three cytokines are derived from the plotted IFN-γ+ and IFN-γ− populations.
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