Novel immunodominant peptide presentation strategy: a featured HLA-A*2402-restricted cytotoxic T-lymphocyte epitope stabilized by intrachain hydrogen bonds from severe acute respiratory syndrome coronavirus nucleocapsid protein

Abstract

Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β(2)-microglobulin (β(2)m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.

Figures

FIG. 1.

HLA class I restriction of the immunogenic region within SARS N protein. (A). The sequence of amino acid residues 330 to 363 along the N protein. N330 to N354 was an immunogenic region within SARS N protein identified in a previous study (21). Peptide N1 (N346 to N354), which is shown in boldface, is included by both peptide NC9585 (N338 to N354) and NC9586 (N346 to N363). (B) Data reanalysis of the ELISPOT assays performed with three HLA-A24+ donors recovered from SARS donors (patient 1, patient 2, and patient 3 in the ID [identification] column in Table 2). The entry 8586 represents the peptide pools NX6, NY7, and NY8, which contain peptides NC9585 and NC9586. Meanwhile, No8586 represents the other peptide pools that do not include these two peptides. The top and bottom of each rectangular box denote the standard error (SE), with the median shown inside the box. Horizontal bars extending from each box represent the 90th and 10th percentiles. Rhombus dots indicate the specific CD8+ T-cell responses of the recovered individual donors stimulated by different peptide pools. One dot represents the average response of one individual donor reacting to one of the peptide pools from two independent tests. The total number of dots for entry 8586 is 9, which was calculated as follows: 3 peptide pools × 3 HLA-A24+ donors. There are 12 pools which do not include these two peptides; therefore, the number of dots for No8586 is 36, which was calculated as follows: 12 peptide pools × 3 HLA-A24+ donors. Details for peptide pools can be found in reference .

FIG. 2.

Binding affinity of peptide N1 to the HLA-A*2402 molecule. (A) In vitro refolding of HLA-A*2402 heavy chain and β2m with N1. As compared to the much lower peak of refolding without any peptide, N1 together with the positive control peptide Nef138-10 could help the HLA-A*2402 complexes refold. The peaks of the complexes with the expected molecular mass of 45 kDa were eluted at the estimated volume of 16 ml on a Superdex 200 column (GE Healthcare). The profile is marked with the approximate positions of the molecular mass standards of 67.0, 35.0, 13.7, and 6.5 kDa. (B) Peptide binding to HLA-A*2402 was quantified by using a T2-A24 stabilization assay. M.F.I., mean fluorescence intensity. These results are representative of three independent experiments.

FIG. 3.

Structure of the HLA-A*2402/N1 complex. (A) Overview of the structure of HLA-A*2402/N1. (B) The electron density of N1 shows the conformation of the peptide with the second residue of N-terminus F2 and the C-terminus residue L9 to anchor in the HLA groove. Shown here are final 2Fo-Fc-stimulated annealing omit maps contoured at 1.0 σ.

FIG. 4.

Conformational features of peptide N1 in the binding groove of HLA-A*2402. (A) D4, N5, and V6 in the middle part of peptide N1 (yellow) bulge out of the peptide binding groove, with the backbone of the three residues rising about 2.3 Å compared to peptide VYG (pink; PDB ID no. 2BCK). (B) The distance between β-carbons of the residues at position 2 and position 9 of N1 (yellow) and VYG (pink) shows the whole length of N1 adopts a more bulged conformation. (C) Two intrachain hydrogen bonds are formed between K3 and N5 and also between N5 and I7, respectively. The side chains of residues D4 and V6, between the two hydrogen bonds, protrude out of the HLA groove and may play a dominant role in the TCR-MHC docking. (D) Central residues of N1 interact with α-helix of HLA-A*2402 through two water molecules.

FIG. 5.

Detection of peptide N1-specific CD8+ T cells in PBMCs of HLA-A24+ donors recovered from SARS by ELISPOT. The mean numbers of SFCs in 105 splenocytes are represented with bars as a measure of IFN-γ secretion from human PBMCs stimulated with peptides. The PBMC samples from two HLA-A24+ donors recovered from SARS were thawed and manipulated in ELISPOT assays.

FIG. 6.

Identification of the immunogenicity of N1 by fluorescence staining and flow cytometry. (A) In tetramer staining, N1-specific CD8+ T cells were measured from N1-stimulated PBMCs of an HLA-A24+ donor recovered from SARS (patient 1) and an HLA-A24+ healthy donor (donor 1), using the PE-labeled HLA-A*2402/N1 tetramer along with PE-Cy5-labeled anti-CD3 and FITC-labeled anti-CD8 MAbs for cell staining. (B) PBMCs from an HLA-A24+ donor recovered from SARS (patient 3) and an HLA-A24+ healthy donor (donor 2) were stained with 1 μM CFSE and stimulated by peptides for 7 days. Panels represent percentages of cells that have undergone divisions.

FIG. 7.

Topology of the newly identified presentation strategy of featured peptides. Featureless peptides have a typical amino acid length (8 to 10 amino acids) but have few or no solvent-exposed residues with prominent side chains (A and D). The previously determined characteristic of featured peptide is defined as type 1 featured peptide (B). These peptides comprise solvent-exposed residues presenting prominent side chains for recognition by a diverse TCR repertoire (C). In this study, a new presentation strategy of featured peptides was identified (E). Peptide without solvent-exposed residue arches itself through intrachain hydrogen bonds and water molecules under the main chain of the peptide. Through this strategy, short side chains of peptide N1 (yellow) protrude to the level of solvent-exposed residues of peptide VYG (pink) and may be recognized by an immune T-cell repertoire of high diversity (F).