Synthetic peptides derived from SARS coronavirus S protein with diagnostic and therapeutic potential - PubMed (original) (raw)

. 2005 Apr 11;579(10):2130-6.

doi: 10.1016/j.febslet.2005.02.070.

Xiao-Dong Wu, Mu De Shi, Rui Fu Yang, You Yu He, Chao Bian, Tie Liu Shi, Sheng Yang, Xue-Liang Zhu, Wei-Hong Jiang, Yi Xue Li, Lin-Chen Yan, Yong Yong Ji, Ying Lin, Guo-Mei Lin, Lin Tian, Jin Wang, Hong Xia Wang, You Hua Xie, Gang Pei, Jia Rui Wu, Bing Sun

Affiliations

Synthetic peptides derived from SARS coronavirus S protein with diagnostic and therapeutic potential

Wei Lu et al. FEBS Lett. 2005.

Abstract

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is an important viral structural protein. Based on bioinformatics analysis, 10 antigenic peptides derived from the S protein sequence were selected and synthesized. The antigenicity and immunoreactivity of all the peptides were tested in vivo and in vitro. Four peptides (P6, P8, P9 and P10) which contain B cell epitopes of the S protein were identified, and P8 peptide was confirmed in vivo to have a potential in serological diagnosis. By using a syncytia formation model, we tested the neutralization ability of all 10 peptides and their corresponding antibodies. It is interesting to find that P8 and P9 peptides inhibited syncytia formation, suggesting that the P8 and P9 spanning regions may provide a good target for anti-SARS-CoV drug design. Our data suggest that we have identified peptides derived from the S protein of SARS-CoV, which are useful for SARS treatment and diagnosis.

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Figures

Figure 1

Figure 1

Spike protein‐ACE2‐mediated syncytia formation model. (A) HEK293T cells were transfected with SARS‐CoV spike protein gene and pUHD15‐1 (SV40); while at the same time, other HEK293T cells were transfected with ACE2 gene and pUHD10‐3. The two kinds of treated cells can fuse with each other and form syncytia, which contain pUHD15‐1 and pUHD10‐3 together and induce the reporter gene expression. (B) The model was tested by a known neutralizing monoclonal antibody, which was obtained from mice immunized with S protein integrated pseudo‐virus. (−) group: ACE2 is not transduced into the HEK293T cells, so no syncytia formation. (+) group: control IgG Ab was used in the test, resulting in the completed syncytia formation.

Figure 2

Figure 2

Map of peptides used for immunization. The spike protein is predicted to be cleaved at amino acid 667 and 668 according to bioinformatics analysis. (A) Full‐length S protein, S1 and S2 fragments were expressed by E. coli and served as target proteins for Western blot. (B) Eight peptide sequences were selected from the S1 fragment and two peptide sequences were selected from the S2 fragment.

Figure 3

Figure 3

Specificity of the peptide‐induced antisera as indicated by Western blot analysis. Lane 1: recombinant SARS‐CoV Spike protein expressed in E. coli; lane 2: S1 fragment expressed in E. coli; lane 3: S2 fragment; lane 4: SARS‐CoV infected Vero E6 cell lysate; lane 5: non‐infected Vero E6 cell lysate. Details are seen in Table 2. Sn, native spike protein; Se, recombinant S protein expressed in E. coli; S1, recombinant S1 fragment of S protein expressed in E. coli; S2, recombinant S2 fragment.

Figure 4

Figure 4

Immunoreactivities of all peptides (P1–P10). The purified IgG from SARS patients’ sera served as S‐protein specific Ab. BSA was used as a negative control. The binding activities of the IgG to all 10 peptides derived from S protein were detected by ELISA. The purified human IgG of SARS patients were 1:500 dilution and HRP‐coupled anti‐human IgG served as second Ab.

Figure 5

Figure 5

The extended PL‐8 peptide showed higher immunoreactivity and higher specificity than P8. (A) The PL‐8 had a higher affinity to the IgG purified from the sera of SARS patients than that of P8. (B) The specificity of peptides to SARS sera was compared between PL8 and IL‐12R peptides. PL8 showed a high specificity and affinity to SARS sera, but it did not response to the sera from health control.

Figure 6

Figure 6

Neutralization test on S‐ACE2 syncytia formation model. (A) Six antisera from P5 to P10, which have good reaction with S proteins, were selected to test their inhibition. Only anti‐P6 and anti‐P8 Abs showed minor inhibition. (−) group: ACE2 is not transduced into the HEK293T cells, so no syncytia formation. (+) group: no Ab was used in the test, result in the completed syncytia formation. a‐N group: antibodies against an irrelative peptide N1 (from N protein of SARS‐CoV) was used in the test as a control. (B) All of the peptides were used in the test, and peptides P8 and P9 can inhibit the syncytia formation to nearly 50%. (−) group: ACE2 was not transduced into the HEK293T cells, so no syncytia formation; (+) group: no peptide was used in the test, resulting in complete syncytia formation; (N) group: an irrelevant peptide N1 (from N protein of SARS‐CoV) was used in the test as a control; no inhibition was observed. (C) Only P8 and P9 peptides were selected to do the test again, and the data demonstrate the inhibition remarkably. All of the experiments were repeated several times. (−) group: ACE2 was not transduced into the HEK293T cells, so no syncytia formation. (+) group: no peptide was used in the test, resulting in complete syncytia formation. (N) group: an irrelevant peptide N1 was used in the test, no inhibition was observed.

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