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. 2021;11(5):1267-1289.
doi: 10.1016/j.jcmgh.2020.12.012. Epub 2021 Jan 11.

Norovirus-Specific CD8+ T Cell Responses in Human Blood and Tissues

Ajinkya Pattekar  1 , Lena S Mayer  2 , Chi Wai Lau  1 , Chengyang Liu  3 , Olesya Palko  4 , Meenakshi Bewtra  5 , Hpap Consortium  6 , Lisa C Lindesmith  7 , Paul D Brewer-Jensen  7 , Ralph S Baric  7 , Michael R Betts  8 , Ali Naji  3 , E John Wherry  9 , Vesselin T Tomov  10
Affiliations

Norovirus-Specific CD8+ T Cell Responses in Human Blood and Tissues

Ajinkya Pattekar et al. Cell Mol Gastroenterol Hepatol. 2021.

Abstract

Background & aims: Noroviruses (NoVs) are the leading cause of acute gastroenteritis worldwide and are associated with significant morbidity and mortality. Moreover, an asymptomatic carrier state can persist following acute infection, promoting NoV spread and evolution. Thus, defining immune correlates of NoV protection and persistence is needed to guide the development of future vaccines and limit viral spread. Whereas antibody responses following NoV infection or vaccination have been studied extensively, cellular immunity has received less attention. Data from the mouse NoV model suggest that T cells are critical for preventing persistence and achieving viral clearance, but little is known about NoV-specific T-cell immunity in humans, particularly at mucosal sites.

Methods: We screened peripheral blood mononuclear cells from 3 volunteers with an overlapping NoV peptide library. We then used HLA-peptide tetramers to track virus-specific CD8+ T cells in peripheral, lymphoid, and intestinal tissues. Tetramer+ cells were further characterized using markers for cellular trafficking, exhaustion, cytotoxicity, and proliferation.

Results: We defined 7 HLA-restricted immunodominant class I epitopes that were highly conserved across pandemic strains from genogroup II.4. NoV-specific CD8+ T cells with central, effector, or tissue-resident memory phenotypes were present at all sites and were especially abundant in the intestinal lamina propria. The properties and differentiation states of tetramer+ cells varied across donors and epitopes.

Conclusions: Our findings are an important step toward defining the breadth, distribution, and properties of human NoV T-cell immunity. Moreover, the molecular tools we have developed can be used to evaluate future vaccines and engineer novel cellular therapeutics.

Keywords: Norovirus T(RM); Norovirus Tetramers; Norovirus-Specific T Cells; T Cell Epitopes.

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Figures

None
Graphical abstract
Figure 1
Figure 1
NoV functional antibodies in healthy donors. Serum from 3 healthy adult donors was assessed using a blockade assay that measures the ability of samples to block interaction of VLPs with ligands. A panel of antigenically diverse GI and GII VLPs representing time-ordered pandemic strains was tested. Donors 1 and 2 were secretors (A and B), whereas Donor 3 was a non-secretor (C). Blockade antibody titers and IC50 values (reported in parentheses as reciprocal of the serum dilution 95% confidence interval) are summarized in (D). Each sample was assayed in 10-fold serial dilution in minimum of 2 independent experiments.
Figure 2
Figure 2
T-cell responses after stimulation with NoV peptide libraries. (A) Three peptide libraries spanning each ORF of the GII.4 2002 Farmington Hills strain were assembled. Each library consisted of 15-mer peptides overlapping neighboring peptides by 10 amino acids. A total of 496 peptides were synthesized. (B) Experimental design to amplify NoV-specific T-cell responses. Donor PBMCs were incubated with all 496 peptides, and responding cells were amplified over 10 days using IL2. Cells were then washed, briefly rested, and re-stimulated with individual libraries, smaller peptide pools, or single candidate peptides in the presence of brefeldin A. IFN-γ and TNF production was assessed by flow cytometry. (C) CD4+ and CD8+ T-cell responses in 3 donors after stimulation with full set of 496 peptides or smaller libraries spanning individual ORFs. Gated on live CD4+ or CD8+ T cells. These experiments were repeated at least 3 times.
Figure 3
Figure 3
Peptide library screening strategy. (A) Overlapping pools of 20–25 peptides were assembled in 3-dimensional matrix arrangement. In this example, peptide 137 was included in pools 1, 7, and 13. (B) Donor PBMCs were stimulated with each of the 15 pools, and cytokine responses were detected with pools 1, 7, and 13, suggesting that peptide 137 contained an immunodominant epitope. (C) PBMCs from the same donor were then stimulated with peptide 137, confirming a robust response. (D) Analysis of peptide 137 using the IEDB (https://www.iedb.org/) uncovered 2 potential HLA-restricted epitopes within the 15-mer sequence that were subsequently tested (Figure 4A).
Figure 4
Figure 4
Defining HLA-restricted immunodominant HLA class I. Candidate 15-mer peptides were identified as described in Figure 3. Next, shorter peptides were generated from 15-mers on the basis of donor HLA types and predicted anchor residues (https://www.iedb.org/ and http://www.cbs.dtu.dk/services/NetMHCpan/). These shorter candidate epitopes were tested using the method shown in Figure 2. Anchor residues are shown in red. (A–C) Epitopes deriving from 15-mer library peptides for Donors 1, 2, and 3. (D) Summary of epitope sequences, location, and HLA restriction.
Figure 5
Figure 5
Detection of Nov-specific CD8+T cells using HLA-peptide tetramers. (A) PBMCs from 3 donors were stained with HLA-matched tetramers to detect NoV-specific CD8+ T cells. Representative of 3 independent experiments. (B) To confirm tetramer specificity, each tetramer was used to stain HLA-matched and HLA-mismatched PBMCs. Total abundance of Tet+ cells for each donor was calculated by adding the percentage of Tet+ cells from HLA-matched samples and subtracting nonspecific staining from HLA-mismatched samples. Donor 1: (0.017 + 0.023 + 0.038) – (0.008 + 0.001 + 0.008) = 0.061. Donor 2: (0.020 + 0.017 + 0.019) – (0.004 + 0.003 + 0.003) = 0.046. (C) PBMCs from Donors 1 and 2 were stimulated for 18 hours with all 496 NoV peptides (without addition of IL2), and IFN-γ and TNF responses were measured by flow cytometry. Unstimulated PBMCs were analyzed in parallel, and the nonspecific cytokine signal was subtracted from stimulated samples. Donor 1: (0.063 – 0.020) = 0.043. Donor 2: (0.063 – 0.012) = 0.051. (D) Summary of data from (B) and (C).
Figure 6
Figure 6
NoV-specific CD8+ T cells are distributed broadly across donors and tissues. (A) PBMCs from 13 additional HLA-typed adult donors were stained with tetramers. (B) Cells from MLN and SPL from a deceased donor (Donor 4) who was an HLA match for epitopes 14, 106, and 32 were stained with tetramers. (C) LPMCs from 2 different deceased donors (Donors 5 and 6) whose HLAs were a match for epitopes 14, 106, and 32 were stained with tetramers. Staining for (B) and (C) could only be done once because of limited samples. Gated on live CD8+ T cells.
Figure 7
Figure 7
Phenotypic characterization of NoV-specific CD8+T cells. (A) PBMCs or (B) LPMCs were stained with tetramers and a panel of antibodies against memory, homing, exhaustion, proliferation, and cytotoxicity markers. Total live CD8+ T cells are shown in grey with Tet+ cells overlayed in red. Representative of 3 independent experiments.
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Comment in

  • Crossing the T's on Norovirus.
    Lee S, Baldridge MT. Lee S, et al. Cell Mol Gastroenterol Hepatol. 2021;11(5):1543-1544. doi: 10.1016/j.jcmgh.2021.01.021. Epub 2021 Feb 19. Cell Mol Gastroenterol Hepatol. 2021. PMID: 33617792 Free PMC article. No abstract available.

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