Cross-reactivity between hepatitis C virus and Influenza A virus determinant-specific cytotoxic T cells

Abstract

The cellular immune response contributes to viral clearance as well as to liver injury in acute and chronic hepatitis C virus (HCV) infection. An immunodominant determinant frequently recognized by liver-infiltrating and circulating CD8(+) T cells of HCV-infected patients is the HCV(NS3-1073) peptide CVNGVCWTV. Using a sensitive in vitro technique with HCV peptides and multiple cytokines, we were able to expand cytotoxic T cells specific for this determinant not only from the blood of 11 of 20 HCV-infected patients (55%) but also from the blood of 9 of 15 HCV-negative blood donors (60%), while a second HCV NS3 determinant was recognized only by HCV-infected patients and not by seronegative controls. The T-cell response of these healthy blood donors was mediated by memory T cells, which cross-reacted with a novel T-cell determinant of the A/PR/8/34 influenza A virus (IV) that is endogenously processed from the neuraminidase (NA) protein. Both the HCV NS3 and the IV NA peptide displayed a high degree of sequence homology, bound to the HLA-A2 molecule with high affinity, and were recognized by cytotoxic T lymphocytes with similar affinity (10(-8) M). Using the HLA-A2-transgenic mouse model, we then demonstrated directly that HCV-specific T cells could be induced in vivo by IV infection. Splenocytes harvested from IV-infected mice at the peak of the primary response (day 7 effector cells) or following complete recovery (day 21 memory cells) recognized the HCV NS3 peptide, lysed peptide-pulsed target cells, and produced gamma interferon. These results exemplify that host responses to an infectious agent are influenced by cross-reactive memory cells induced by past exposure to heterologous viruses, which could have important consequences for vaccine development.

FIG. 1

HCV_NS3-1073-specific cytotoxicity is mediated by cells expanded from the CD45RO⁺ memory T-cell pool.

FIG. 2

MHC binding affinity. TAP-deficient T2 cells were cultured for 16 h at 26°C to enhance expression of peptide-receptive cell surface molecules and then incubated with various concentrations of individual peptides at 37°C for 2 h, washed, and stained with fluorescein-conjugated anti-HLA-A2 antibody (One Lambda Inc.) and 1 μg of propidium iodide per ml. Data express the mean fluorescence intensity of live, propidium iodide-negative cells.

FIG. 3

(A) IFN-γ production assessed by direct ex vivo Elispot analysis of PBMC from healthy, HCV-negative blood donors. Blood donors were divided into two groups, those with (left panel) and without (right panel) HCV_NS3-1073-specific CTLs. PBMC from each individual were then tested directly ex vivo for production of IFN-γ during a 30-h incubation with the indicated peptide. Responses were considered positive if more than 10 specific spots were detected and if the number of spots in the presence of antigen was at least twofold greater than the number of spots in the absence of antigen. (B) IFN-γ production assessed by direct ex vivo Elispot analysis of PBMC from HCV-infected patients. HCV-infected patients were divided into two groups, those with (left panel) and without (right panel) HCV_NS3-1073-specific CTLs. PBMC from each individual were then tested directly ex vivo for production of IFN-γ during a 30-h incubation with the indicated peptide. Responses were considered positive if more than 10 specific spots were detected and if the number of spots in the presence of antigen was at least twofold greater than the number of spots in the absence of antigen.

FIG. 4

In vitro cross-reactivity. Cryopreserved PBMC of five HCV-seronegative, healthy controls were stimulated for 3 weeks in vitro in the presence of IL-2, IL-7, IL-12, and 10 μg of IV_NA-231, HPV_L1-315, or WAG₆₁ peptide per ml. Each CTL line was tested in a 6-h ⁵¹Cr release assay against C1R-A2 targets pulsed overnight with 10 μg of HCV_NS3-1073 (▪), IV_NA-231 (▨), HPV_L1-315 (░⃞), or WAG1₆₁ (□) per ml. The 10% cutoff for a positive CTL assay is indicated by the horizontal line.

FIG. 5

T-cell receptor affinity. PBMC of patient Chr-2 were stimulated for 3 weeks in the presence of IL-7, IL-12, and 10 μg of peptide IV_NA-231 (A) or peptide HCV_NS3-1073 (B) per ml. CTL lines were tested in a 6-h ⁵¹Cr release assay against target cells pulsed with the indicated concentrations of HCV_NS3-1073 or IV_NA-231 peptide.

FIG. 6

(A) HCV_NS3-1073-specific CTLs recognize IV-infected target cells. IV PR8-infected C1R-A2 cells (filled circles) or uninfected C1R-A2 cells (open circles) were labeled with ⁵¹Cr for 1 h and used as target cells in a standard 6-h ⁵¹Cr release assay with the HCV_NS3-1073-specific CTL line derived from patient Chr-5. (B) IV_NA-231-specific CTL lines recognize IV-infected target cells. IV PR8-infected C1R-A2 cells (filled squares) or uninfected C1R-A2 cells (open squares) were labeled with ⁵¹Cr for 1 h and used as target cells in a standard 6-h ⁵¹Cr release assay with IV_NA-231-specific CTL effectors derived from healthy, HCV-negative blood donor HD-7. (C) Cytotoxic activity of an HCV_NS3-1073-specific CTL line from patient Chr-5 against peptide-pulsed or IV-infected target cells. The specific lysis at an effector/target ratio of 33:1 is shown.

FIG. 7

Induction of HCV_NS3-1073-specific CTL by IV infection in vivo. HLA-A2-transgenic mice were infected intraperitoneally with ≈500 hemagglutinating units of PR8 IV, 10⁷ PFU of wild-type WR strain VV (Wt-VV), or 10⁷ PFU of recombinant VV expressing HCV-NS3 (NS3-VV). At 21 days following immunization, splenocytes were stimulated in vitro for 7 days in the presence of 10 μg of the indicated peptides per ml. (A) IFN-γ production as assessed by Elispot analysis. (B) Cytotoxicity tested in a standard 6-h ⁵¹Cr release assay. Cytotoxicity was tested against peptide-coated and noncoated target cells; the specific cytotoxicity, i.e., cytotoxicity in the presence of peptide minus cytotoxicity in the absence of peptide, is shown. Similar results for both IFN-γ production and the ⁵¹Cr release assay were observed for splenocytes harvested 7 days after infection.