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. 2002 Sep 2;196(5):579-88.
doi: 10.1084/jem.20011255.

Long-lived memory T lymphocyte responses after hantavirus infection

Heather L Van Epps  1 , Masanori Terajima, Jukka Mustonen, T Petteri Arstila, Elizabeth A Corey, Antti Vaheri, Francis A Ennis
Affiliations

Long-lived memory T lymphocyte responses after hantavirus infection

Heather L Van Epps et al. J Exp Med. .

Abstract

Puumala virus (PUUV) is a hantavirus that causes hemorrhagic fever with renal syndrome (HFRS), which is an important public health problem in large parts of Europe. We examined the memory cytolytic T lymphocyte (CTL) responses in 13 Finnish individuals who had HFRS between 1984 and 1995. In seven of these donors, we detected virus-specific CTL responses against the PUUV nucleocapsid (N) protein after in vitro stimulation with PUUV. Six novel CD8(+) CTL epitopes were defined on the N protein and were found to be restricted by various HLA alleles including A2, A28, B7, and B8. This is the first demonstration of PUUV-specific CTL responses in humans, and the first identification of CTL epitopes on PUUV. In addition, this study provides one of the few characterizations of a human antiviral memory T cell response, without the complicating issues of virus persistence or reinfection. Interferon (IFN)-gamma ELISPOT analysis showed that memory CTL specific for these epitopes were present at high frequency in PUUV-immune individuals many years after acute infection in the absence of detectable viral RNA. The frequencies of PUUV-specific CTL were comparable to or exceeded those found in other viral systems including influenza, EBV and HIV, in which CTL responses may be boosted by periodic reinfection or virus persistence.

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Figures

Figure 1.
Figure 1.
Bulk culture recognition of PUUV proteins. PBMCs (4–5 × 106) were stimulated with 20 μl of infectious PUUV (strain K27; 2.6 × 106 pfu/ml) and cytotoxicity assays were performed using autologous BLCLs infected with recombinant vaccinia viruses expressing PUUV N, G1, G2/Bs or G2/Sm protein as targets. The recombinant vaccinia virus designated vac-G2/Bs contains the first half of the G2 cDNA (amino acids 1 to 256), and the recombinant designated vac-G2/Sm contains the second half of the G2 protein (amino acids 227 to 490). E/T ratios were either 60:1 or 80:1. Unlabeled wild-type vaccinia virus infected BLCL were included in all wells at a ratio of 10:1 unlabeled: labeled targets, to reduce levels of vaccinia virus-specific lysis. Data shown represent the percent specific lysis after subtraction of wild-type vaccinia virus background lysis. Responses >10% above background were considered positive.
Figure 2.
Figure 2.
CTL activity of PUUV N-specific CD8+ T cell clones isolated from donor 1 against autologous BLCL target cells pulsed with increasing dilutions of the optimal peptide. E/T = 10. (A) CTL lines specific for the A2-restricted epitope N204–12. (B) CTL lines specific for the B7,B8-restricted epitope N173–81. (C) CTL line specific for the epitope N236–50. Lysis of targets not pulsed with peptide was <5% in all assays.
Figure 3.
Figure 3.
Detection of virus-specific CD8+ memory T cells by IFN-γ ELISPOT. PBMCs were stimulated with 10 μg/ml of the indicated peptides in a 16–18 h assay. Input cell numbers ranged from 2–4 × 106 cells per well. Peptide stimulations were performed in triplicate wells. PBMCs stimulated with a B8-restricted epitope from HCV NS3 (NS31402–1411) was included for comparison with PUUV-specific B8-restricted epitopes. PBMCs incubated with media alone was included as negative controls. The data is presented as the number of IFN-γ producing cells/106 PBMCs, with media control values subtracted. The number of spots in the media control wells ranged from 0 to 5. All ELISPOT experiments were performed at least twice and mean values are shown. (A) PUUV-immune individuals who had high precursor frequencies (>100 specific T cells/106 PBMC) of CD8+ T cells specific for one or more CD8+ epitopes on PUUV N. (B) PUUV-immune individuals who had lower precursor frequencies of CD8+ T cells specific for one or more CD8+ epitopes on PUUV N.
Figure 3.
Figure 3.
Detection of virus-specific CD8+ memory T cells by IFN-γ ELISPOT. PBMCs were stimulated with 10 μg/ml of the indicated peptides in a 16–18 h assay. Input cell numbers ranged from 2–4 × 106 cells per well. Peptide stimulations were performed in triplicate wells. PBMCs stimulated with a B8-restricted epitope from HCV NS3 (NS31402–1411) was included for comparison with PUUV-specific B8-restricted epitopes. PBMCs incubated with media alone was included as negative controls. The data is presented as the number of IFN-γ producing cells/106 PBMCs, with media control values subtracted. The number of spots in the media control wells ranged from 0 to 5. All ELISPOT experiments were performed at least twice and mean values are shown. (A) PUUV-immune individuals who had high precursor frequencies (>100 specific T cells/106 PBMC) of CD8+ T cells specific for one or more CD8+ epitopes on PUUV N. (B) PUUV-immune individuals who had lower precursor frequencies of CD8+ T cells specific for one or more CD8+ epitopes on PUUV N.
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