key: cord-0958559-xys0g8b8
authors: Shichijo, S.; Keicho, N.; Long, H.T.; Quy, T.; Phi, N.C.; Ha, L.D.; Ban, V.V.; Itoyama, S.; Hu, C.‐J.; Komatsu, N.; Kirikae, T.; Kirikae, F.; Shirasawa, S.; Kaji, M.; Fukuda, T.; Sata, M.; Kuratsuji, T.; Itoh, K.; Sasazuki, T.
title: Assessment of synthetic peptides of severe acute respiratory syndrome coronavirus recognized by long‐lasting immunity
date: 2004-10-20
journal: Tissue Antigens
DOI: 10.1111/j.1399-0039.2004.00314.x
sha: 14eebf1dcb0a236f7414ed9c75755bafc181739f
doc_id: 958559
cord_uid: xys0g8b8

Abstract: In order to determine highly immunogenic severe acute respiratory syndrome coronavirus (SARS‐CoV) epitope peptides capable of inducing long‐lasting immunity, we first screened immunoglobulin‐G (IgG) antibodies reactive to 197 different overlapping 15‐mers from the SARS‐CoV proteins in the sera of three infected patients. Forty‐two peptides among them were reactive to the sera from all three patients. Consequently, we tested for the reactivity of these 42 peptides to patients' sera (n = 45) at 6‐month post‐infection. The significantly higher levels of IgG antibodies specific to three (S791, M207 and N161) of 42 peptides were detectable in the post‐infection sera from 23 (51%), 27 (60%) and 19 (42%) of 45 patients, respectively. These three peptides, recognized by their long‐lasting immunity, may provide a better understanding of the immunogenicity of SARS‐CoV.

. Each peptide was dissolved in dimethylsulfoxide (DMSO) and was then stored at À20 C until use. These peptides were tested for their reactivity to the sera of early stages of three Taiwanese SARS-CoV-infected patients by using flowmetry analysis with Luminex TM (Luminex Corp., Austin, TX) (9) . The sera were collected from Jen-Ai Municipal Hospital, SaAn District, Taipei, Taiwan. The patients' sera showed significantly higher levels of immunoglobulin-G (IgG) (P < 0.05) activities reactive to 42 of 197 peptides tested, including 20 spike (S)-, seven membrane (M)-and 15 nucleocapsid (N)-derived peptides, when the means of the scores of fluorescence intensity (FI) from the sera (1000-fold dilution) of the three patients (closed bar) were compared to those of the three healthy donors (HD) (open bar). The peptides were coupled to colour-coded beads, according to the modified manufacturer's instructions (Luminex Corp.). In brief, 100 ml of colour-coded beads were mixed with 100 ml of peptide (1 mg/ml in 0.1 M morpholinoehanesulfonic acid (MES) buffer, pH 4.5). The peptide-loaded beads were then incubated with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide 2-(N-Morpholino)ethanesulfonic acid (EDC) (1 mg ml À1 ) at room temperature for 30 min in darkness, and the beads were washed with Tween-20 phosphatebuffered saline (PBS). The beads were treated with 2-aminoethanol for 10 min at room temperature in darkness, washed twice and then re-suspended with 1 ml of 0.05% Block Ace (Snow Brand Milk Products Co., Ltd, Hokkaido, Japan) in Tween-20 PBS. Two microlitres of serum at dilutions of 100-10,000 times was incubated with 25 ml of the peptide-coupled colour-coded beads for 2 h at room temperature on a plate shaker in a 96-well filter plate (MultiScreen TM -BV, Millipore Co., Bedford, MA). After incubation, the plate was washed by using a vacuum manifold apparatus and was incubated with 100 ml of biotinylated goat anti-human IgG (gamma-chain-specific: Vector Laboratory Inc., Burlingame, CA) for 1 h at room temperature on a plate shaker. The plate was then washed, and 100 ml of streptavidin-PE (Molecular Probes, Eugene, OR) was added into wells, followed by incubation for 30 min at room temperature on a plate shaker. The bound beads were washed three times followed by the addition of 100 ml of Tween-20 PBS into each well, and the plate was placed for 3 min on a plate shaker. Fifty microlitres of sample was analysed by using the Luminex TM system with the help of the method reported previously (13, 9, 15) . (Table 1 ). In contrast, there were no significant differences in the reactivity against any of 42 peptides between the HD and the contact persons.

Anti-N161 Anti-S791 (Table 1) . However, the positive cases showing FI scores of greater than the mean plus 2SD were only six of 45 patients (13%). In contrast to these four peptides, significant levels of IgG reactive to the remaining 36 peptides were either scarcely or not detected in the patients (Table 1) Fig. 5 . Absorption test. The immunoglobulin-G (IgG) activity to each of the S791, M207 and N161 peptides was absorbed by using a triplicate assay with an immobilized corresponding peptide and each of the five different irrelevant peptides. The method for the preparation of immobilized peptides was the same as the method used for ELISA plate preparation, as described in the legend of Fig. 4 . The results of the absorption test were analysed by means of a two-tailed Student's t-test. All tests of significance were two-sided. In order to test the specificity of anti-peptide IgG in the serum samples, 100 ml/well of serum samples (1 : 100 dilution with 0.05% PBST) was absorbed with the immobilized peptide (200 mg/well: closed bar or 40 mg/well: open bar, as final concentrations) in wells kept for 2 h at room temperature. The absorption was repeated three times, and then the level of peptide-specific IgG in the resultant supernatant was measured. PBST, Tween-20 PBS.

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The authors thank Dr Nguyen Le Hang, Ms. Nguyen Thu Ha and Pham Phuong Thuy for supporting the co-ordination and implementation of this research project in Vietnam, and Drs Masamichi Koujiro and Akira Yamada of Kurume University School of Medicine, Asahi-machi, Kurume, Fukuoka, Japan, for co-ordinating this research.