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. 2020 Feb 3;221(4):578-588.
doi: 10.1093/infdis/jiz491.

Generation of Norovirus-Specific T Cells From Human Donors With Extensive Cross-Reactivity to Variant Sequences: Implications for Immunotherapy

Ryo Hanajiri  1 , Gelina M Sani  1 , Devin Saunders  1 , Patrick J Hanley  1   2   3 , Abha Chopra  4   5 , Simon A Mallal  4   5 , Stanislav V Sosnovtsev  6 , Jeffrey I Cohen  6 , Kim Y Green  6 , Catherine M Bollard  1   2   3 , Michael D Keller  1   2   7
Affiliations

Generation of Norovirus-Specific T Cells From Human Donors With Extensive Cross-Reactivity to Variant Sequences: Implications for Immunotherapy

Ryo Hanajiri et al. J Infect Dis. .

Abstract

Background: Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences.

Methods: Norovirus-specific T cells were generated from peripheral blood of healthy donors by stimulation with overlapping peptide libraries spanning the entire coding sequence of the norovirus genome.

Results: We successfully generated T cells targeting multiple norovirus antigens with a mean 4.2 ± 0.5-fold expansion after 10 days. Norovirus-specific T cells comprised both CD4+ and CD8+ T cells that expressed markers for central memory and effector memory phenotype with minimal expression of coinhibitory molecules, and they were polyfunctional based on cytokine production. We identified novel CD4- and CD8-restricted immunodominant epitopes within NS6 and VP1 antigens. Furthermore, NSTs showed a high degree of cross-reactivity to multiple variant epitopes from clinical isolates.

Conclusions: Our findings identify immunodominant human norovirus T-cell epitopes and demonstrate that it is feasible to generate potent NSTs from third-party donors for use in antiviral immunotherapy.

Keywords: T cells; adoptive immunotherapy; norovirus; primary immunodeficiency; transplantation.

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Figures

Figure 1.
Figure 1.
Norovirus-specific T-cells can be generated from peripheral blood of norovirus-seropositive donors. (A) Peripheral blood mononuclear cells from healthy donors were stimulated with norovirus pepmixes on day 0. Fold expansion was measured 10 days after stimulation based on absolute cell counting in total cell numbers (n = 20, mean ± standard error of the mean [SEM]). (B) Phenotype of the expanded cells was accessed by flow cytometry for T-cell markers (CD3, CD4, and CD8), natural killer (NK) cell markers (CD16), and memory subsets (CD45RO and CD62L) (n = 9, mean ± SEM). (C) Specificity of the expanded cells with response to norovirus antigens stimulation was assayed by interferon (IFN)-γ enzyme-linked immunospot (ELISpot) assay (n = 20, mean ± SEM). Unstimulated T cells (CTL only) and stimulation with actin were used as negative controls. Results are presented as spot-forming cells (SFC)/1 × 105 cells. The number of spots were compared with those from actin control (*, P < .05 and **, P < .01; 2-tailed Student’s t test). (D) Specificity of the expanded cells against norovirus antigens between norovirus-seropositive donors (n = 17) and seronegative donors (n = 3) by IFN-γ ELISpot (mean ± SEM). CM, central memory T-cells (CD45RO+CD62L+); EM, effector memory T-cells (CD45RO+CD62L−); NKs, natural killer cells (CD3−CD16+); NKTs, natural killer T cells (CD3+CD16+); Treg, regulatory T cells (CD3+CD4+CD25+CD127dim).
Figure 2.
Figure 2.
Norovirus-specific T-cells are polyfunctional. Norovirus-specific T cells were tested for secretion of proinflammatory cytokines (A) and regulatory cytokines (B) in response to stimulation with pooled pepmixes. Cytokines were measured in cell supernatant after 24-hour stimulation by Luminex assay. Stimulation with actin was used as a control (n = 7, mean ± standard error of the mean [SEM]). (C) Polycytokine (interferon [IFN]-γ and tumor necrosis factor [TNF]-α) production in response to pooled norovirus pepmixes as evaluated by intracellular cytokine staining from CD4+ and CD8+ T cells in 1 representative donor (gated on CD3+). Stimulation with actin was used as a negative control. (D) Summary results of double cytokine (IFN-γ and TNF-α) producing cells in CD4+ and CD8+ T-cell populations (n = 6, mean ± SEM). (E) Expression of coinhibitory markers of norovirus-specific T cells by CD4+ and CD8+ T cells after restimulation with pooled norovirus pepmixes (n = 9, mean ± SEM). GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin.
Figure 3.
Figure 3.
Identification of VP1 CD8-restricted T-cell epitopes. T-cell epitope mapping for VP1 protein was performed, and results from 1 representative donor are shown. (A) The breadth of T-cell reactivity was evaluated using a total of 21 mini-peptide pools, each containing 8–12 peptides, spanning the entire VP1 protein. Mini-pools were made in such a way that each peptide was present in 2 pools. A norovirus-specific T-cell product was stimulated with 21 mini-peptide pools, and the T-cell responses were measured by interferon (IFN)-γ enzyme-linked immunospot (ELISpot). Responses to pools 3 and 10, 3 and 15, 3 and 16, 3 and 19, 3 and 21, 4 and 10, 4 and 15, 4 and 16, 4 and 19, 4 and 21, 7 and 10, 7 and 15, 7 and 16, 7 and 19, and 7 and 21 may be induced by individual 15-mer peptides 363, 368, 369, 372, 374, 375, 380, 381, 384, 386, 411, 416, 417, 420, and 422, respectively (yellow cells). (B) Testing of these single peptides by IFN-γ ELISpot revealed recognition of the single 15-mer peptides of 374, 375, 380, 381, 386, and 420 (mean ± standard error of the mean [SEM]). (C) Intracellular IFN-γ staining was used to evaluate the human leukocyte antigen (HLA)-restriction of the single 15-mer peptides (gated on CD3+). Results indicating CD8-restricted epitopes are shown. (D) Blocking experiments with HLA class I and II blocking antibodies by IFN-γ ELISpot (mean ± SEM). (E) To identify the minimal 9-mer epitope, recognition against a panel of 9-mers overlapping by 8 amino acids spanning the sequence of 420 was evaluated by IFN-γ ELISpot (mean ± SEM). (F) Restricted HLA allele was evaluated by IFN-γ ELISpot using allogeneic HLA partially matched phytohemagglutinin (PHA) blasts alone or pulsed with GRNTHNVHL (GRN) peptide (mean ± SEM). Autologous PHA blasts pulsed with GRN were used as a positive control. SFC, spot-forming cells.
Figure 4.
Figure 4.
Cross-reactivity of norovirus-specific T cells to variant epitopes. Norovirus-specific T cells were stimulated with CD8-restricted T-cell epitopes GRNTHNVHL (A), YVVIGVHTA (B), FPGEQLLFF (C), and CD4-restricted T-cell epitope PIKQIQIHKSGEFCR (D), as well as altered versions as identified in database or clinical isolates. Responses were measured by interferon (IFN)-γ enzyme-linked immunospot (ELISpot) (mean ± standard error of the mean [SEM]). (E) Cross-reactivity of norovirus-specific T cells against variant epitopes was measured by IFN-γ ELISpot assay. Data are cross-reactivity indices (variant response/GII.4 Sydney 2012 response) of 45 NS6 (n = 9) and 26 VP1 epitopes (n = 11) (mean ± SEM). SFC, spot-forming cells.
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