Department of Biotechnology, College of Engineering, Yonsei University, 134 Shinchon-Dong, Seodaemun-Gu, Seoul 120-749, South Korea1
Mogam Biotech. Institute, 341 Pojung-ri, Koosung-myun, Yongin-city Kyonggi-do 449-910, South Korea2
Author for correspondence: Baik Seong. Fax +82 2 362 7265. e-mail blseong{at}yonsei.ac.kr
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Abstract |
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In this study, we investigated whether virus-specific CTLs, especially recognizing the epitope conserved in both HTNV and SNV, are generated in HTNV-infected patients. So far, it is believed that several MHC haplotypes, such as HLA-B8, DR3 and HLA-B35, play an important role in the cellular immune response in the pathology of both hantavirus pulmonary syndrome (HPS) and HFRS (Koster et al., 1998 ; Van Epps et al., 1999
; Ennis et al., 1997
). And yet, human HLA-A2.1-restricted CTL epitopes have not been confirmed clearly. Here, we identified HLA-A2.1-restricted epitopes conserved in both HTNV and SNV and showed that some CD8+ T cells in bulk-cultured PBMCs derived from HTNV-infected patients strongly recognized epitope peptides.
To identify conserved epitopes, we performed amino acid sequence alignments between HTNV (strains 76-118, C1-1 and C1-2) and SNV (strains NMH10 and NMR11). Amino acid sequences were obtained from the NCBI database. Using the computer program BIMAS (NIH) (Parker et al., 1994 ), nine CTL epitopes, conserved in the nucleocapsid (N) and polymerase (P) proteins of both viruses, were selected based on their binding scores, as calculated by the program. The sequence of each peptide and its position in the viral genome are summarized in Table 1
, along with their relative solubility in buffer.
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Among the nine different peptides tested, six peptides from HTNV (aa 334342, 512520, 559567, 628636, 11881196 and 12731281) showed significant binding affinities (Fig. 1). In this study, the hepatitis B virus S antigen (HBs) peptide, aa 271280 (Lee et al., 1997
), was used as a positive control for the class I MHC stabilization assay with T2 cells. With peptide concentrations of 10 µg/ml, surface HLA-A2 expression on T2 cells reached a plateau, indicating that the concentration is sufficient for the saturation of surface expression of HLA-A2.1 molecules in T2 cells. The P9 peptide derived from HTNV, aa 17361744 (SNV, aa 17371745), exhibited higher affinity relative to any of the other peptides tested. Peptides P1, -5, -7 and -8 were found to be moderate binders, whereas peptide P2 was found to bind relatively weakly (Fig. 1
). Peptides P3 --4 and -6 did not show significant binding under our experimental setting. These results suggested that, although the strength of HLA-A2.1 binding was variable, six of nine peptides exhibited significant binding affinities and, therefore, are expected to be immunogens for generating antigen-specific cytotoxic T cells in HTNV-infected patients.
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Peptide P1 (HTNV, aa 334342; SNV, aa 333341) elicited the strongest CTL activity among the various peptides tested. To identify its specific CTL more closely, CD8+ T cells were purified from PBMCs using magnetic beads and characterized with regard to surface markers, profiles of secreted cytokines and MHC-restriction. Purified CD8+ cells were repeatedly stimulated with the peptide-pulsed allogenic HLA-A2.1-positive PBMCs as feeder cells in order to enrich antigen-specific CTLs against the peptide. After 28 days, cells were harvested, stained with FITC-conjugated anti-CD8 antibody and PE-conjugated anti-CD4, -CD25, -CD45RO, -CD45RA, -CD62 or -CD69 antibodies and analysed by flow cytometry. It has been reported that, in activated or effector CD8+ T cells, CD25, CD45RO and CD69 molecules are strongly increased, whereas CD45RA and CD62 molecules are significantly down-regulated as compared to naive CD8+ T cells (Detels et al., 1996 ; Rutella et al., 1998
). Consistent with previous reports, CD25, CD45RO and CD69 molecules were strongly increased in purified CD8+ cells, whereas no significant increases in CD45RA and CD62 molecules were observed (Fig. 3b
) as compared to the pre-cultured total PBMCs derived from the same patient (Fig. 3a
).
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Next, a chromium-release assay was used to test whether or not these cells show antigenic specificity against target cells pulsed with peptide P1. As shown in Fig. 3(d,
), specific lysis of target cells pulsed with P1 was significantly high, up to about 78% at an effector:target cell ratio of 25:1, as compared to the negative controls pulsed with an irrelevant peptide (HBs, aa 271280) (Fig. 3d
,
) or without peptide (Fig. 3d
,
). These results confirm that the majority of CD8+ cells are restricted by HLA-A2.1 molecules pulsed with HTNV peptide P1. Although partially cloned T cells generated in this study may not represent a homogeneous clone, their properties were very similar to that of a cloned T cell in terms of antigen-specific recognition, effector-dependence in target cell killing, phenotype and cytokine profiles.
In the present study, we predicted and selected cytotoxic T cell epitopes conserved in both HTNV and SNV proteins and examined antigen-specific responses from HTNV-infected patients. Five of seven patients exhibited significant activity against six different epitopes. Furthermore, the peptide ILQDMRNTI (HTNV, aa 334342; SNV, aa 333341) was shown to elicit strong CTL activity in five of seven patients tested. Following prolonged stimulation with the peptide, activated and MHC-restricted CD8+ cells producing Tc type 1 cytokines could be detected in the PBMCs from HTNV-infected patients, strongly suggesting that the epitope would be potentially useful for the development of prophylactic or therapeutic vaccines for both HTNV and SNV infections.
A key issue is whether homologous peptides derived from the two related viruses are also immunogenic in SNV-infected patients. Since we did not have access to PBMCs from SNV-infected patients in our present circumstances, we opted to test cytolytic activity with PBMCs from HTNV-infected patients. Although the pathogenesis of HTNV and SNV is not well understood, it has been proposed that they might induce a similar immune response in infected patients (Lopez et al., 1996 ; Nichol et al., 1993
; Williams et al., 1997
). Since the CTL epitopes tested in this study are also present in SNV proteins as well, it is probable that specific responses in SNV infection might also be generated. In conclusion, we have identified several HLA-A2.1-restricted CTL epitopes conserved in both HTNV and SNV. These epitopes may be used as useful components for peptide-based, DNA or chimeric viral vector vaccines for the intervention of both HTNV and SNV infections.
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Acknowledgments |
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References |
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Received 20 November 2001;
accepted 7 January 2002.