title: Residue analysis of a CTL epitope of SARS-CoV spike protein by IFN-gamma production and bioinformatics prediction.
abstract: BACKGROUND
  Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by the novel coronavirus SARS-CoV. The T cell epitopes of the SARS CoV spike protein are well known, but no systematic evaluation of the functional and structural roles of each residue has been reported for these antigenic epitopes. Analysis of the functional importance of side-chains by mutational study may exaggerate the effect by imposing a structural disturbance or an unusual steric, electrostatic or hydrophobic interaction.
  
  
  RESULTS
  We demonstrated that N50 could induce significant IFN-gamma response from SARS-CoV S DNA immunized mice splenocytes by the means of ELISA, ELISPOT and FACS. Moreover, S366-374 was predicted to be an optimal epitope by bioinformatics tools: ANN, SMM, ARB and BIMAS, and confirmed by IFN-gamma response induced by a series of S358-374-derived peptides. Furthermore, each of S366-374 was replaced by alanine (A), lysine (K) or aspartic acid (D), respectively. ANN was used to estimate the binding affinity of single S366-374 mutants to H-2 Kd. Y367 and L374 were predicated to possess the most important role in peptide binding. Additionally, these one residue mutated peptides were synthesized, and IFN-gamma production induced by G368, V369, A371, T372 and K373 mutated S366-374 were decreased obviously.
  
  
  CONCLUSIONS
  We demonstrated that S366-374 is an optimal H-2 Kd CTL epitope in the SARS CoV S protein. Moreover, Y367, S370, and L374 are anchors in the epitope, while C366, G368, V369, A371, T372, and K373 may directly interact with TCR on the surface of CD8-T cells.
authors.affiliation: Institute of Immunology, Zhongshan School of Medicine, Key Laboratory of Tropical Disease Control Research of Ministry of Education, Sun Yat-sen University, Guangzhou, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Huang
authors.forename: Yingnan
authors.initials: Y
authors.lastname: Cao
authors.forename: Xianzhang
authors.initials: X
authors.lastname: Bu
authors.forename: Changyou
authors.initials: C
authors.lastname: Wu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019295:Computational Biology; D058501:Enzyme-Linked Immunospot Assay; D018604:Epitope Mapping / Q000379:methods; D018984:Epitopes, T-Lymphocyte / Q000276:immunology / Q000378:metabolism*; D005260:Female; D007371:Interferon-gamma / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D045473:Severe acute respiratory syndrome-related coronavirus / Q000276:immunology*; D020539:Sequence Analysis, Protein; D045169:Severe Acute Respiratory Syndrome / Q000276:immunology*; D064370:Spike Glycoprotein, Coronavirus; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000276:immunology / Q000378:metabolism*
chemical_list: D018984:Epitopes, T-Lymphocyte; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; C578557:spike glycoprotein, SARS-CoV; C578553:spike protein, mouse hepatitis virus; D007371:Interferon-gamma