Identification of H-2Kb-restricted T-cell epitopes within the nucleocapsid protein of Hantaan virus and establishment of cytotoxic T-cell clonesThis work was performed at Korea Advanced Institute of Science and Technology and Mogam Biotech Research Institute.

Abstract

Although neutralizing antibodies against Hantaan virus (HTV) can protect hosts from viral infection, T-cell responses to HTV are also important in host defense against HTV. However, much less is known about cytotoxic T lymphocyte (CTL) responses to HTV. To identify CTL epitopes in the HTV nucleocapsid protein (NP), we selected 7 H-2K(b)-motif-fitting peptides. Of these peptides, 3 peptides (NP3, NP4, and NP7) were recognized by CTL responses derived from HTV-immunized mouse splenocytes. NP3 and NP4 peptides were also recognized by HTV-immunized splenocytes after secondary in vitro stimulation with the relevant peptide, but NP7 could not be recognized after in vitro stimulation. These results agree well with peptide immunization studies showing that peptide-specific CTL responses could be induced with NP3 and NP4 but not with NP7 peptide. Furthermore, CTL activity assay using targets, prepared to express the antigen (NP) endogenously, demonstrated that NP3 and NP4 peptides could be presented endogenously. CTL elicited with NP4 peptide retained some cross-reactivity and was difficult to long-term culture. However, NP3-elicited CTL was very specific for NP3 peptide and was stable enough to be cloned. Among many CTL lines elicited with HTV or HTV NP peptides, 6 NP3-specific CTL clones were established and have been maintained more than 2 years. All 6 CTL clones were characterized to be CD3+, CD4-, CD8+, CD25+, CD62L-, and NK1.1-, and to use TCR Vbeta6. This preferential usage of TCR Vbeta6 indicates that TCR Vbeta6 regions are important for recognition of the HTV NP3 epitope (NP221-228, SVIGFLAL) on H-2K(b) molecule. Our data demonstrate the definition of mouse CTL epitopes in HTV and the generation of HTV-specific mouse CTL clones.

Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus

“…We selected and synthesized 15 peptides and tested their respective antigenicity according to binding score. A higher score means a stronger bind to MHC (Park et al, 2000). This first crucial step increases the probability of successful epitope selection.…”

“…Each peptide is H-2K b -restricted. HTNV nucleoprotein (NP) aa221–aa228 (SVIGFLAL) derived from the HTNV nucleoprotein was used as a positive control epitope (Park et al, 2000). We used computer algorithms in Immune Epitope Database and Analysis Resources (IEDB-http://tools.iedb.org/main/) to identify optimal peptide epitopes within the reactive 8-mer peptides.…”

“…Cells with nonspecific phytohemagglutinin stimulation (PHA, 10 μg/mL, Sigma-Aldrich, St. Louis, MO) or without peptide stimulation served as positive and negative controls, respectively. Additionally, spleen cells pulsed with the immunodominant NP221–228 peptide (Park et al, 2000) was used as an epitope positive control. For quantification of ex vivo responses, the assay was performed in duplicate.…”