BACKGROUND
severe acute respiratory syndrome , a newly emerging infectious disease, is caused by a sars-associated coronavirus   <cit> , which may originate from some wild animals  <cit> .
after its first occurrence, sars rapidly spread around the world along international air-travel routes, reaching all five continents and resulting in several hundreds of deaths  <cit> .
the most recent epidemic of sars occurred in beijing and anhui, china in april  <dig> and originated from laboratory contamination .
although the outbreaks seem to be over, sars remains a safety concern because of the possible reintroduction of a sars-like coronavirus  into humans and the risk of an escape of sars-cov from laboratories  <cit> .
more importantly, a new recombinant virus derived from human  <cit> , swine and/or avian influenza virus, might re-emerge as a new sars-cov type, much like the recent emergence of a novel swine-origin influenza a  in humans.
thus, it is essential to develop various and distinct strategies to combat this highly contagious disease.
the published literature reports that high titres of neutralizing antibodies and sars-cov-specific cytotoxic t lymphocyte  responses were detected in patients who had recovered from sars  <cit> , and the levels of those responses correlated well with disease outcome  <cit> .
hence, both humoral and cellular immune responses appear to be crucial for the clearance of sars-cov infection.
immune responses can be raised directly against several of the sars-cov proteins  <cit> .
targeting the spike  structural glycoprotein  <cit>  in particular induces a robust immune response, suggesting it plays an important role in the systemic clearance of sars-cov  <cit> .
the viral surface s protein is involved in host cell receptor recognition, virus attachment and entry  <cit> ; adaptive evolution of s protein, thus, contributes to sars-cov overcoming the species barrier  <cit> .
hence, many vaccines and therapeutics against sars-cov target the s protein  <cit> .
considering that cytotoxic t-cell responses participate in the clearance of virus from recovered sars patients and contribute to immunopathology in early stages of the disease  <cit> , one of the most attractive s protein-based strategies proposes eliciting a sars-cov ctl response to clear the infection.
to this end, a detailed understanding of the s protein-mediated ctl response is essential.
development of effective treatments and vaccines against sars-cov depends upon the underlying mechanisms of various immune effectors in protective immunity and identification of the protective antigens recognized by each.
epitopes are the basic antigenic elements of virus structural proteins, which functionally induce the host cell-mediated immune response.
identification of the ctl-specific epitopes of sars-cov proteins could provide the basis for the development of sars immunity-based treatments and aid in the understanding of mechanisms underlying sars-cov pathogenesis.
hla-a* <dig> is expressed 39-46% of all major ethnicities  <cit> .
the identification of hla-a*0201-restricted sars-cov/s ctl epitopes is an important contribution towards understanding the role of ctls in sars-cov pathogenesis and protection.
currently, several ctl-specific sars-cov s protein epitopes have been identified in the context of hla-a* <dig>  including s411- <dig>  s787- <dig>  s978- <dig>  s1042- <dig>  s1167- <dig>  s1203- <dig> and ssp- <dig>  <cit> , and the h <dig> complex, including s366- <dig>  s436- <dig>  s525- <dig> and s1031- <dig>  <cit> .
it is likely that additional s protein ctl epitopes exist.
s protein is relatively large in size and usage of different detection methods may result in the identification of novel s protein-derived epitopes.
in turn, this data will provide advances towards understanding the mechanisms of sars-cov infection, and contribute to the development of future sars-cov infection intervention strategies.
in this study, we identified a novel sars/s-specific, hla-a*0201-restricted epitope that was conserved among sars-cov strains.
based on a binding affinity-based prediction and a proteosomal cleavage site prediction, we constructed a panel of potential hla-a*0201-restricted ctl peptides from the s protein.
each candidate peptide was evaluated for its binding affinity to hla-a* <dig> molecules using the t <dig> cell-peptide binding test.
we then evaluated the ability of hla-a* <dig> binding peptides to provoke ctl responses in peripheral blood lymphocytes .
pbl preparations from major histocompatibility complex -matched healthy donors or hla-a <dig> /kb transgenic  mice, were incubated with dendritic cells  that had been pre-pulsed with the peptides of interest.
we identified a novel sars-cov s protein-derived ctl epitope s that was capable of priming the s protein-specific hla-a <dig> -restricted ctl response.
the effective ctl response was evidenced by cell death of peptide-pulsed t <dig> and peptide-pulsed jurkat-a2/kb cells.
the findings of this study should provide insight into the immunological characteristics of spike protein and provide an alternative strategy for the future development of sars-cov s protein ctl epitope-based vaccines.
RESULTS
selection of potential hla-a* <dig> binding peptides derived from sars-cov/s protein
we selected candidate ctl epitopes derived from sars-cov s protein by two criteria:  conservation between different strains of sars-cov to encompass as many sars-cov strains as possible, and  high representation in the general population, i.e., hla-a*0201-restricted.
after alignment of the amino acid residues of s protein with eighteen sars-cov strains , the bj <dig> strain s protein was selected to predict the s protein specific, hla-a*0201-restricted ctl epitopes.
based on the presence of hla-a* <dig> binding motifs and the cleavage sites for proteasomes and immunoproteasomes, eight candidate peptides were predicted and synthesized , termed sp1- <dig>  these peptides were further verified as having an absence of shared sequence homology with the human or murine proteins using the blast search engine http://www.ncbi.nlm.nih.gov/blast/, to avoid any autoreactive response.
* hla-a*0201-binding motif score from algorithm syfpeithi ≥  <dig>  the eight peptides were also predicted and selected using propred <dig>  in which the threshold of hla-a2-binding motif is 4% and the threshold of proteasomal and immunoproteasomal cleavage site is 8%.
† increase of hla-a* <dig> molecules on t <dig> cell surface.
fi = .
fi >  <dig>  indicates high-affinity peptides; fi ≤  <dig> , low-affinity peptides.
hla-a*0201-restricted peptide s411- <dig> was used as a positive control for hla-a*0201-binding ability, while the h-2b-restricted peptide hbcag was used as a negative control.
to evaluate the binding affinity of these peptides to hla-a* <dig> molecules, a t <dig> cell-peptide binding test was used  <cit> .
t <dig> cells lack the transporter associated with antigen processing , a key factor involved in endogenous antigen processing and presentation, causing the empty hla-i molecules on the t <dig> cell surface to be very unstable and to degrade rapidly after cell surface presentation.
however, when exogenous epitope peptides bind to the hla-i molecules on the cell surface, they become stable  <cit> .
accordingly, the peptide-induced upregulation of hla-i on tap-deficient t <dig> cells is used to monitor peptide binding to class i molecules, which then indicates the binding affinity of peptides to hla-i molecules.
higher-affinity peptides will induce more hla-a* <dig> expression on the cell surface than will lower-affinity peptides.
as shown in table  <dig>  of the eight candidate peptides only sp <dig>  sp <dig>  sp <dig> and sp <dig> were high-affinity epitopes .
the positive control peptide, s411- <dig>  bound hla-a* <dig> strongly , whereas no binding was observed with the negative control hbcag peptide .
ex vivo generation of peptide-specific cd8+ ctls from healthy human donor pbls
to investigate the capacity of candidate peptides to mobilize a human ctl repertoire, hla-a2+ pbls from ten hla-a2+ donors were stimulated in vitro by dcs loaded with the eight candidate peptides, positive control peptides s411- <dig>  or negative control peptides hbcag.
t <dig> cells loaded with each of peptides were used as target cells in cytotoxicity assays.
of the eight peptides tested, sp <dig>  sp <dig> and sp <dig> induced more cd8+ t-cells that specifically produced ifn-γ in response to dcs pulsed with the relevant peptides or positive control peptide in comparison to other groups .
furthermore, these cd8+ t cells could lyse the t <dig> cells loaded with relevant peptides or positive control peptide more efficiently than other groups .
the cytolysis observed were specific because the ctls could not lyse t <dig> cells loaded with irrelevant peptides .
due to the limited induction of ifn-γ secreting t cell frequency and the low ctl cytolysis ability, groups other than sp <dig>  sp <dig> and sp <dig> were excluded from further study.
the ex vivo results showed the existence of functional anti-sars-cov/s ctl precursors in the peripheral t cell repertoire of healthy donors.
furthermore, sars-cov/s-derived peptides sp <dig>  sp <dig> and sp <dig> could not only induce the increased s protein specific ifn-γ secreting t cell frequency but also the enhanced cytolytic capacity of these ctls.
in vivo induction of peptide-specific cd8+ ctls in hla-a <dig> /kb transgenic mice
to further address whether the immunogenic candidate peptide is naturally processed and presented, hla-a <dig> /kb transgenic mice were immunized with s/pvax <dig> plasmid containing a full-length cdna encoding the sars-cov/s protein.
splenocytes were collected from mice seven days after four weekly injections with s/pvax <dig>  and re-stimulated ex vivo by mouse bone marrow-derived dcs loaded with the candidate peptides, the positive peptide s411- <dig>  the irrelevant peptides hbcag, or dcs alone, for an additional  <dig> days.
investigation of ifn-γ production and the cytolytic ability of the effector ctl cells were carried out following the re-stimulation.
the j cells loaded with the corresponding peptides were used as targets in cytotoxicity assays.
as shown in figure 3a, ctls from s/pvax1-immunized mice were able to lyse three candidate peptides-pulsed j cells but did not lyse j cells alone or j cells loaded with irrelevant peptide hbcag at any e/t ratio.
of the three candidate peptides tested, sp <dig> exhibited the most lytic capacity at each e/t ratio, which was comparable to the positive control peptide .
in accordance with results from the cytolytic assays, bulk ctls released ifn-γ only in response to dcs pulsed with sp <dig>  sp <dig>  sp <dig> and the positive control peptide, but not to those pulsed with irrelevant peptide hbcag or dcs alone .
again, among the three tested candidate peptides, sp <dig> released the most ifn-γ following peptide stimulation .
discussion
it is known that sars-cov can induce a strong specific ctl response in infected patients, besides high titres of neutralizing antibodies  <cit> .
furthermore, the ctl response levels correlate with disease outcome  <cit> , suggesting ctl response is crucial for the clearance of sars-cov.
among all the encoded proteins in the sars-cov genome, s protein is currently considered the most important target to prime the host immune response  <cit> .
it has been reported that an inflammatory cell influx of airway macrophages and a massive release of cytokines occur during the peak of sars infection  <cit> .
thus, it is reasonable to investigate the underlying mechanism of specific ctl response induced by sars-cov s protein during sars-cov infection which may have a positive role in sars-cov clearance or a negative role in sars-cov immunopathogenesis.
in this study, we predicted and validated a novel ctl epitope of sars-cov s protein.
we used two prediction systems to identify candidate ctl epitopes of s protein  to improve prediction accuracy.
the eight predicted peptides were then verified via mhc peptide binding assay .
among the eight candidate peptides, sp <dig>  sp <dig>  sp <dig> and sp <dig> exhibited the highest capacity to induce more potent ctls secreting ifn-γ and to lyse target cells from hla-a*0201-matched healthy donor pbls .
further in vivo investigation showed that plasmid encoding the full-length sars-cov s gene elicited strong ctl response in hla-a <dig> /kb transgenic mice.
these ctls could produce substantial amounts of ifn-γ and kill target cells in a peptide-specific and hla-a*0201-restricted manner , suggesting the predicted candidate peptides were native epitopes, capable of priming ctl responses in vivo.
we found that candidate peptide sp <dig> held the greatest ability to secrete ifn-γ and kill target cells in vivo .
another candidate peptide, sp <dig>  failed to induce the most potent peptide specific ctls in tg mice , despite it having had the highest such ability in comparison to the rest of the in vitro stimulation set , indicating the need for candidate peptides to be biofunctionally validated in vivo.
to date, several ctl epitopes of sars-cov s protein have been identified in the context of hla-a* <dig>  including s411- <dig>  s787- <dig>  s978- <dig>  s1042- <dig>  s1167- <dig>  s1203- <dig> and ssp- <dig>  <cit> , or of the h <dig> complex, including s366- <dig>  s436- <dig>  s525- <dig> and s1031- <dig>  <cit> .
in this study, we predicted and validated a novel ctl epitope of sars-cov s protein, sp <dig> .
this may be due to the unique predictive methods used in our study.
we combined strategies for prediction .
previous studies used single methods, such as hla peptide binding prediction or overlapping peptide strategy  <cit> , suggesting different prediction strategies might lead to different results.
in any case, the predicted candidate peptides require additional validation methods to ensure accuracy.
in our study, we also determined that among the eight peptides we predicted, four could potent prime ctls to produce significant ifn-γ and lyse target cells; although, sp <dig> peptide exhibited the most potency for ctl priming.
however, zhou et al.
reported that they only found one predicted peptide that could stimulate ifn-γ secretion and target cell lysis  <cit> .
this may reflect the different stimulators used in these studies; zhou used peptides to stimulate the effector cells directly while we used dcs loaded with the candidate peptides.
we argue for the use of dcs as stimulator cells in ex vivo study because dcs are the most potent apcs for priming t cells, and they not only present peptides to t cells but also provide many important co-stimulatory signals.
moreover, under in vivo conditions dcs present peptides to t cells in the context of mhc molecules.
thus, using dcs as ex vivo stimulator cells most closely mimics the in vivo context.
CONCLUSIONS
our study has identified a novel conserved hla-a*0201-restricted epitope from the spike protein of sars-cov.
we propose that the newly identified epitope could be used for evaluation of sars-cov-specific cd8+ t-cell responses during the course of sars infection and treatment.
this epitope should also aid in the characterization of virus control mechanisms and immunopathology of sars-cov infection.
ultimately, our findings may be relevant to the development of ethnically unbiased, widely applicable immunotherapeutic approaches for sars disease.

 severe acute respiratory syndrome, a newly emerging infectious disease, is caused by a sars-associated coronavirus, which may originate from some wild animals. after its first occurrence, sars rapidly spread around the world along international air-travel routes, reaching all five continents and resulting in several hundreds of deaths. the most recent epidemic of sars occurred in beijing and anhui, china in april and originated from laboratory contamination. although the outbreaks seem to be over, sars remains a safety concern because of the possible reintroduction of a sars-like coronavirus into humans and the risk of an escape of sars-cov from laboratories. more importantly, a new recombinant virus derived from human, swine and/or avian influenza virus, might re-emerge as a new sars-cov type, much like the recent emergence of a novel swine-origin influenza a in humans. thus, it is essential to develop various and distinct strategies to combat this highly contagious disease. the published literature reports that high titres of neutralizing antibodies and sars-cov-specific cytotoxic t lymphocyte responses were detected in patients who had recovered from sars, and the levels of those responses correlated well with disease outcome. hence, both humoral and cellular immune responses appear to be crucial for the clearance of sars-cov infection. immune responses can be raised directly against several of the sars-cov proteins. targeting the spike structural glycoprotein in particular induces a robust immune response, suggesting it plays an important role in the systemic clearance of sars-cov. the viral surface s protein is involved in host cell receptor recognition, virus attachment and entry; adaptive evolution of s protein, thus, contributes to sars-cov overcoming the species barrier. hence, many vaccines and therapeutics against sars-cov target the s protein. considering that cytotoxic t-cell responses participate in the clearance of virus from recovered sars patients and contribute to immunopathology in early stages of the disease, one of the most attractive s protein-based strategies proposes eliciting a sars-cov ctl response to clear the infection. to this end, a detailed understanding of the s protein-mediated ctl response is essential. development of effective treatments and vaccines against sars-cov depends upon the underlying mechanisms of various immune effectors in protective immunity and identification of the protective antigens recognized by each. epitopes are the basic antigenic elements of virus structural proteins, which functionally induce the host cell-mediated immune response. identification of the ctl-specific epitopes of sars-cov proteins could provide the basis for the development of sars immunity-based treatments and aid in the understanding of mechanisms underlying sars-cov pathogenesis. hla-a* is expressed 39-46% of all major ethnicities. the identification of hla-a*0201-restricted sars-cov/s ctl epitopes is an important contribution towards understanding the role of ctls in sars-cov pathogenesis and protection. currently, several ctl-specific sars-cov s protein epitopes have been identified in the context of hla-a* including s411- s787- s978- s1042- s1167- s1203- and ssp-, and the h complex, including s366- s436- s525- and s1031-. it is likely that additional s protein ctl epitopes exist. s protein is relatively large in size and usage of different detection methods may result in the identification of novel s protein-derived epitopes. in turn, this data will provide advances towards understanding the mechanisms of sars-cov infection, and contribute to the development of future sars-cov infection intervention strategies. in this study, we identified a novel sars/s-specific, hla-a*0201-restricted epitope that was conserved among sars-cov strains. based on a binding affinity-based prediction and a proteosomal cleavage site prediction, we constructed a panel of potential hla-a*0201-restricted ctl peptides from the s protein. each candidate peptide was evaluated for its binding affinity to hla-a* molecules using the t cell-peptide binding test. we then evaluated the ability of hla-a* binding peptides to provoke ctl responses in peripheral blood lymphocytes. pbl preparations from major histocompatibility complex -matched healthy donors or hla-a /kb transgenic mice, were incubated with dendritic cells that had been pre-pulsed with the peptides of interest. we identified a novel sars-cov s protein-derived ctl epitope s that was capable of priming the s protein-specific hla-a -restricted ctl response. the effective ctl response was evidenced by cell death of peptide-pulsed t and peptide-pulsed jurkat-a2/kb cells. the findings of this study should provide insight into the immunological characteristics of spike protein and provide an alternative strategy for the future development of sars-cov s protein ctl epitope-based vaccines.  selection of potential hla-a* binding peptides derived from sars-cov/s protein we selected candidate ctl epitopes derived from sars-cov s protein by two criteria: conservation between different strains of sars-cov to encompass as many sars-cov strains as possible, and high representation in the general population, i.e., hla-a*0201-restricted. after alignment of the amino acid residues of s protein with eighteen sars-cov strains, the bj strain s protein was selected to predict the s protein specific, hla-a*0201-restricted ctl epitopes. based on the presence of hla-a* binding motifs and the cleavage sites for proteasomes and immunoproteasomes, eight candidate peptides were predicted and synthesized, termed sp1- these peptides were further verified as having an absence of shared sequence homology with the human or murine proteins using the blast search engine http://www.ncbi.nlm.nih.gov/blast/, to avoid any autoreactive response. * hla-a*0201-binding motif score from algorithm syfpeithi ≥ the eight peptides were also predicted and selected using propred in which the threshold of hla-a2-binding motif is 4% and the threshold of proteasomal and immunoproteasomal cleavage site is 8%. † increase of hla-a* molecules on t cell surface. fi =. fi > indicates high-affinity peptides; fi ≤, low-affinity peptides. hla-a*0201-restricted peptide s411- was used as a positive control for hla-a*0201-binding ability, while the h-2b-restricted peptide hbcag was used as a negative control. to evaluate the binding affinity of these peptides to hla-a* molecules, a t cell-peptide binding test was used. t cells lack the transporter associated with antigen processing, a key factor involved in endogenous antigen processing and presentation, causing the empty hla-i molecules on the t cell surface to be very unstable and to degrade rapidly after cell surface presentation. however, when exogenous epitope peptides bind to the hla-i molecules on the cell surface, they become stable. accordingly, the peptide-induced upregulation of hla-i on tap-deficient t cells is used to monitor peptide binding to class i molecules, which then indicates the binding affinity of peptides to hla-i molecules. higher-affinity peptides will induce more hla-a* expression on the cell surface than will lower-affinity peptides. as shown in table of the eight candidate peptides only sp sp sp and sp were high-affinity epitopes. the positive control peptide, s411- bound hla-a* strongly, whereas no binding was observed with the negative control hbcag peptide. ex vivo generation of peptide-specific cd8+ ctls from healthy human donor pbls to investigate the capacity of candidate peptides to mobilize a human ctl repertoire, hla-a2+ pbls from ten hla-a2+ donors were stimulated in vitro by dcs loaded with the eight candidate peptides, positive control peptides s411- or negative control peptides hbcag. t cells loaded with each of peptides were used as target cells in cytotoxicity assays. of the eight peptides tested, sp sp and sp induced more cd8+ t-cells that specifically produced ifn-γ in response to dcs pulsed with the relevant peptides or positive control peptide in comparison to other groups. furthermore, these cd8+ t cells could lyse the t cells loaded with relevant peptides or positive control peptide more efficiently than other groups. the cytolysis observed were specific because the ctls could not lyse t cells loaded with irrelevant peptides. due to the limited induction of ifn-γ secreting t cell frequency and the low ctl cytolysis ability, groups other than sp sp and sp were excluded from further study. the ex vivo results showed the existence of functional anti-sars-cov/s ctl precursors in the peripheral t cell repertoire of healthy donors. furthermore, sars-cov/s-derived peptides sp sp and sp could not only induce the increased s protein specific ifn-γ secreting t cell frequency but also the enhanced cytolytic capacity of these ctls. in vivo induction of peptide-specific cd8+ ctls in hla-a /kb transgenic mice to further address whether the immunogenic candidate peptide is naturally processed and presented, hla-a /kb transgenic mice were immunized with s/pvax plasmid containing a full-length cdna encoding the sars-cov/s protein. splenocytes were collected from mice seven days after four weekly injections with s/pvax and re-stimulated ex vivo by mouse bone marrow-derived dcs loaded with the candidate peptides, the positive peptide s411- the irrelevant peptides hbcag, or dcs alone, for an additional days. investigation of ifn-γ production and the cytolytic ability of the effector ctl cells were carried out following the re-stimulation. the j cells loaded with the corresponding peptides were used as targets in cytotoxicity assays. as shown in figure 3a, ctls from s/pvax1-immunized mice were able to lyse three candidate peptides-pulsed j cells but did not lyse j cells alone or j cells loaded with irrelevant peptide hbcag at any e/t ratio. of the three candidate peptides tested, sp exhibited the most lytic capacity at each e/t ratio, which was comparable to the positive control peptide. in accordance with results from the cytolytic assays, bulk ctls released ifn-γ only in response to dcs pulsed with sp sp sp and the positive control peptide, but not to those pulsed with irrelevant peptide hbcag or dcs alone. again, among the three tested candidate peptides, sp released the most ifn-γ following peptide stimulation. discussion it is known that sars-cov can induce a strong specific ctl response in infected patients, besides high titres of neutralizing antibodies. furthermore, the ctl response levels correlate with disease outcome, suggesting ctl response is crucial for the clearance of sars-cov. among all the encoded proteins in the sars-cov genome, s protein is currently considered the most important target to prime the host immune response. it has been reported that an inflammatory cell influx of airway macrophages and a massive release of cytokines occur during the peak of sars infection. thus, it is reasonable to investigate the underlying mechanism of specific ctl response induced by sars-cov s protein during sars-cov infection which may have a positive role in sars-cov clearance or a negative role in sars-cov immunopathogenesis. in this study, we predicted and validated a novel ctl epitope of sars-cov s protein. we used two prediction systems to identify candidate ctl epitopes of s protein to improve prediction accuracy. the eight predicted peptides were then verified via mhc peptide binding assay. among the eight candidate peptides, sp sp sp and sp exhibited the highest capacity to induce more potent ctls secreting ifn-γ and to lyse target cells from hla-a*0201-matched healthy donor pbls. further in vivo investigation showed that plasmid encoding the full-length sars-cov s gene elicited strong ctl response in hla-a /kb transgenic mice. these ctls could produce substantial amounts of ifn-γ and kill target cells in a peptide-specific and hla-a*0201-restricted manner, suggesting the predicted candidate peptides were native epitopes, capable of priming ctl responses in vivo. we found that candidate peptide sp held the greatest ability to secrete ifn-γ and kill target cells in vivo. another candidate peptide, sp failed to induce the most potent peptide specific ctls in tg mice, despite it having had the highest such ability in comparison to the rest of the in vitro stimulation set, indicating the need for candidate peptides to be biofunctionally validated in vivo. to date, several ctl epitopes of sars-cov s protein have been identified in the context of hla-a* including s411- s787- s978- s1042- s1167- s1203- and ssp-, or of the h complex, including s366- s436- s525- and s1031-. in this study, we predicted and validated a novel ctl epitope of sars-cov s protein, sp. this may be due to the unique predictive methods used in our study. we combined strategies for prediction. previous studies used single methods, such as hla peptide binding prediction or overlapping peptide strategy, suggesting different prediction strategies might lead to different results. in any case, the predicted candidate peptides require additional validation methods to ensure accuracy. in our study, we also determined that among the eight peptides we predicted, four could potent prime ctls to produce significant ifn-γ and lyse target cells; although, sp peptide exhibited the most potency for ctl priming. however, zhou et al. reported that they only found one predicted peptide that could stimulate ifn-γ secretion and target cell lysis. this may reflect the different stimulators used in these studies; zhou used peptides to stimulate the effector cells directly while we used dcs loaded with the candidate peptides. we argue for the use of dcs as stimulator cells in ex vivo study because dcs are the most potent apcs for priming t cells, and they not only present peptides to t cells but also provide many important co-stimulatory signals. moreover, under in vivo conditions dcs present peptides to t cells in the context of mhc molecules. thus, using dcs as ex vivo stimulator cells most closely mimics the in vivo context.  our study has identified a novel conserved hla-a*0201-restricted epitope from the spike protein of sars-cov. we propose that the newly identified epitope could be used for evaluation of sars-cov-specific cd8+ t-cell responses during the course of sars infection and treatment. this epitope should also aid in the characterization of virus control mechanisms and immunopathology of sars-cov infection. ultimately, our findings may be relevant to the development of ethnically unbiased, widely applicable immunotherapeutic approaches for sars disease. 
the spike protein is a major structural glycoprotein of coronavirus, the causal agent of severe acute respiratory syndrome. the s protein is a potent target for sars-specific cell-mediated immune responses. here, we describe a study that leads to the identification of a novel hla-a*0201-restricted epitope from conserved regions of s protein. first, different sars-cov sequences were analyzed to predict eight candidate peptides from conserved regions of the s protein based upon hla-a* binding and proteosomal cleavage. among the four candidate peptides, sp induced specific ctls both ex vivo in pbls of healthy hla-a2+ donors and in hla-a /kb transgenic mice immunized with a plasmid encoding full-length s protein. these results suggest that sp is a naturally processed epitope. four of eight candidate peptides were tested by hla-a* binding assays. we propose that sp epitope should help in the characterization of mechanisms of virus control and immunopathology in sars-cov infection. the immunized mice released ifn-γ and lysed target cells upon stimulation with sp peptide-pulsed autologous dendritic cells in comparison to other candidates.
 the spike protein is a major structural glycoprotein of coronavirus, the causal agent of severe acute respiratory syndrome. the s protein is a potent target for sars-specific cell-mediated immune responses. however, the mechanism cov pathogenesis in sars and the role of special ctls in virus clearance are still largely uncharacterized. here, we describe a study that leads to the identification of a novel hla-a*0201-restricted epitope from conserved regions of s protein.  first, different sars-cov sequences were analyzed to predict eight candidate peptides from conserved regions of the s protein based upon hla-a* binding and proteosomal cleavage. four of eight candidate peptides were tested by hla-a* binding assays. among the four candidate peptides, sp induced specific ctls both ex vivo in pbls of healthy hla-a2+ donors and in hla-a /kb transgenic mice immunized with a plasmid encoding full-length s protein. the immunized mice released ifn-γ and lysed target cells upon stimulation with sp peptide-pulsed autologous dendritic cells in comparison to other candidates.  these results suggest that sp is a naturally processed epitope. we propose that sp epitope should help in the characterization of mechanisms of virus control and immunopathology in sars-cov infection. 