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1、Rev. Med. Virol. 2006; 16: 117131.Published online in Wiley InterScience ().Reviews in Medical VirologyDOI: 10.1002/rmv.492Severe acute respiratory syndrome (SARS)coronavirus: application of monoclonalantibodies and developmentof an effective vaccineYasuko Tsunetsugu-Yokota, Kazuo Ohnishi and Toshit
2、ada Takemori*Department of Immunology, National Institute of Infectious Diseases, Toyama-cho, Shinjuku-ku,Tokyo, JapanSUMMARYSARS-CoV is a new type of human coronavirus identified as a causative agent of severe acute respiratory syndrome(SARS). On the occasion of the SARS outbreak, various monoclona
3、l antibodies (mAbs) against SARS-CoV have beendeveloped and applied for diagnosis, clinical management and basic research. In this review, we overview thebiochemical and functional properties and applications of these SARS-CoV mAbs. We also focus on a variety ofvaccines currently under development a
4、nd discuss the immune response elicited by these vaccines in animal models,hopefully to better understand what we need to do next to fight against newly emerging pathogens in the future.Copyright # 2006 John Wiley & Sons, Ltd.Received: 25 November 2005; Revised: 13 December 2005; Accepted: 13 Decemb
5、er 2005INTRODUCTIONThe outbreak of fatal severe acute respiratory syn-drome (SARS), which originally occurred in 2002 inChina and subsequently spread to many countriesin early 2003, is now under control due to a con-certed world-wide effort led by the World HealthOrganisation(WHO).Acausativepathogen
6、,SARS-CoV, was identified and the entire genomesequence was quickly determined 1,2. SARS-CoV belongs to a distinct group of known humancoronaviruses, such as HCoV-229E (Group 1) andHCoV-OC43 (Group 2), which are only slightlypathogenic in humans. On the contrary, animalcoronaviruses have been known
7、to cause clinicallyserious diseases in livestock and pet animals.Based on the nucleotide sequence homology ofSARS-CoV-like virus in wild animals traded inChinese markets, it is considered that SARS-CoVemerged through the interspecies transmissionof such animal coronaviruses (review in 3). Arecent re
8、port strongly suggested that the Chinesehorseshoe bat was a natural host of SARS-CoV(bat-SARS-CoV) and that caged animals such asHimalayan palm civets and raccoon dogs are theamplification hosts 4.SARS-CoV is readily transmissible through closecontact between family members, and health-careand labor
9、atory workers. Since SARS-CoV infects toand replicates in the respiratory and intestinaltissues, a vaccine eliciting a mucosal neutralisingantibody,particularlyinthebroncho-alveolarlumen would be most desirable. It has beenreported that high titres of neutralising IgG antibo-dies against SARS-CoV ar
10、e present in SARSpatient 5 and that passive administration of ser-um antibodies from previously infected patientsimproved the conditions in newly infected recipi-ents 6. Furthermore, in the mouse model, thepassive transfer of mouse immune serum againstSARS-CoVhasbeendemonstratedtoreducepulmonary vir
11、al titres in mice infected withSARS-CoV 7. These results support the notionthat the neutralising antibodies are crucial forprotection against SARS-CoV infection.On the occasion of the SARS outbreak, severallaboratories endeavoured to identify the mono-clonal antibodies (mAbs) that act against SARS-C
12、oV in order to provide tools for diagnosis, inR R E E V I I E WR R E E V I I E WCopyright # 2006 John Wiley & Sons, Ltd.*Corresponding author: Toshitada Takemori, Department of Immu-nology, National Institute of Infectious Diseases, 1-23-1 Toyama-cho, Shinjuku-ku, Tokyo 162-8640, Japan.E-mail: ttosh
13、inih.go.jpcombination with viral detection by RT-PCR ana-lysis, and for application in the clinical treatment ofSARS patients. In this context, we overview thebiochemical and functional properties of SARS-CoV mAbs, as established by different technicalapproaches, together with the applications which
14、utilise these mAbs in both clinical and basicresearch. We also discuss the immune response eli-cited by a variety of vaccines in animal models tobetter understand what is required for developingan effective vaccine against SARS-CoV infection.MONOCLONAL ANTIBODIESAGAINST SARS-CoVThe SARS-CoV encodes
15、four major structural pro-teins; the spike (S), membrane (M), envelope (E)and nucleocapsid (N) proteins. Up to now, a vari-ety of mouse and human monoclonal antibodies(mAbs) recognising these proteins have beenestablished and are listed in Table 1, for mouse,and Table 2 for human mAbs. In addition,
16、thefine epitope-mapping studies of these mAbs aresummarised in Figure 1. We now know that theS protein binds to the cellular receptor ACE2 8and that ACE2 recognises the S-protein at the ami-no acid residues aa318510 9. This region isdesignated as a receptor binding domain (RBD).Thus, the S protein,
17、especially RBD, is a majortarget for eliciting neutralising antibody. We willfocus on this issue in the next section.Mouse mAbs (Table 1)When whole virions were used as an antigen,established mAbs frequently recognised the S pro-tein. We used a highly purified whole virions ofSARS-CoV as an immunisi
18、ng antigen, which hadbeeninactivatedbyUV-irradiationtoavoidunwanted denaturation of virus protein 10. Themajority of mAbs established (26 out of 29) recog-nised conformational epitopes on the S proteinwith virus-neutralising ability in vitro, whereas3mAbs recognised linear epitopes on the N pro-tein
19、. On the other hand, Chou et al. establishedseveral mAbs which recognise the linear epitopesof S protein by using detergent-disrupted virionsas an antigen 11. Such mAbs, designated 1A5and 2C5, had high neutralisation ability, suggest-ing that neutralising activity is mediated by a vari-ety of antibo
20、dy recognition sites on the S protein.Gubbins et al. used whole virions inactivated with?-propiolactone and obtained 9mAbs with virusneutralising ability 12. Surprisingly, the majorityof mAbs are encoded by the VH-gene family,VHJ558, coupled with variety of light chains, sug-gesting a correlation be
21、tween neutralisation abilityand specific VH-gene allele.Table 1. Establishment of mouse monoclonal antibodiesHostAntigen (adjuvant)ClonesApplicationReference1Whole virion26 clones of anti-S andSandwich-ELISA, Western-blot10UV-inactivated (FCA)3 clones of anti-NHistology, IFA, neutralization(SKOT-8,
22、?9 etc.)2Whole virion7 clones of anti-SEpitope mapping on S,86detergent-inactivated(1A5, 2C5 etc.)Neutralization(FCA)Western-blot3Whole virion5 clones to SMAbs belong to87beta-propiolactone-(F26G18, F26G19 etc.)single VH-gene familyinactivated (FCA)4Recombinant N9 clones to anti-rNSandwich-ELISA, We
23、stern-blot88(MPLTDM)a(NE4A4, NE8A11, etc.)5Recombinant S4 clones to SNeutralization, Western-blot,13fragment (FCA)(S26, S34, S84, S78)ELISA, cyto-staining6RBP-Fcb27 RBP-specific mAbsEpitope mapping14(4D5, 17H9 etc.)NeutralizationaMPL, monophosphoryl lipid A; TDM, trehalose dicorynomycolate.bRBP-Fc,
24、fusion protein containing the receptor binding domain (RBP) linked to a human IgG1 Fc fragment.118118Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.118118Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.Copyright # 2006 John Wiley & Sons, Ltd.Rev. Med. Virol. 2006; 16: 117131.Another approac
25、h is to use the recombinantSARS-CoV proteins as an immunising antigen.Zhou et al. prepared a recombinant protein frag-ment of S protein, S-II (aa 485625), which was pre-dicted to include the RBD 13. This recombinantprotein fragment binds to the surface of Vero cells,and the mAbs against this fragmen
26、t had potentin vitro neutralisation ability. He et al. used a smallfragment of recombinant RBD, which was fusedwith human IgG1 Fc fragment (RBD-Fc) as animmunising antigen 14. Twenty-seven hybrido-mas were established and their antigen specifici-ties were mapped into 6 different conformation-depende
27、nt and 2 adjacent linear epitopes (seebelow) 14. Using recombinant N protein, Cheet al. isolated anti-N mAbs, useful for the sensitiveantigen-capture ELISA system and Western blot15.Human mAbs (Table 2)Considering the clinical approach to block SARS-CoV infection by utilising neutralising antibodies
28、,human mAbs have been established by differenttechniques. Traggiai et al. developed a uniquemethod of EBV transformation of B cells, whichenabled rapid generation of human neutralisingantibodies against SARS-CoV 16. The memoryB cell pools of recovered SARS patients were effi-ciently immortalised, an
29、d high affinity (10?8M to10?11M) human mAbs were successfully obtained.In this case, about 80% of EBV-transformed B cellclones secreted IgG antibody against S proteinand about 15% against N protein, most likelyreflecting the actual memory B cell repertoire inthose individuals who recovered from SARS
30、-CoVinfection.Sui et al. obtained eight clones of two single-chain variable antibody fragments (scFvs) byscreening the scFv library constructed from non-immune human antibodies on the S1 fragment ofSARS-CoV S protein 17. Among these clones,80R bound to the S protein RBD region andshowed an efficient
31、 neutralisation ability 17.Using a similar approach, van den Brink et al.Table 2. Human monoclonal antibodies for SARS-CoVMethodClonesNotesReferencesImproved EBV-transformation ofmemory B cells from patientsanti-S, -N, -E (manyclones, S3.1,S102.1 etc.)In vitro neutralizationactivity, 10?810?11MIn vi
32、vo protection in mousemodel48scFva, phage display screening onrecombinant S1 protein8 scFv clones(80R etc.)Memory B cell repertoirerepresentationEpitope mapping,in vitro neutralization35In vivo protection in mousemodel89S1-genotyping co-related with80R sensitivityscFv, phage display screening onirra
33、diated SARS-CoV wholevirionanti-S, -N (CR3014,CR3018, CR3009)Epitope mapping,immunoelectron microscopy,IFA, in vitro neutralization36scFv, the library constructed fromSARS convalescent patients,selection against inactivatedwhole virionanti-S2 (B1 etc.)Mapped to 10231189 of S2protein, potent neutrali
34、zationactivity in vitro19Transgenic mice with humanimmunoglobulin gene (Medarex)immunized with recombinantS protein2 clones (68, 201)Epitope mapping,In vivo protectionin mouse model21ascFv; single-chain variable antibody fragments.Severe acute respiratory syndrome coronavirusSevere acute respiratory
35、 syndrome coronavirus119119Severe acute respiratory syndrome coronavirusSevere acute respiratory syndrome coronavirus119119Copyright # 2006 John Wiley & Sons, Ltd.Rev. Med. Virol. 2006; 16: 117131.obtained eight scFv clones, of which two clonescould be mapped to the N protein and four tothe S protei
36、n 18. One of the two anti-N scFvclones recognises a linear epitope and all of theothers recognise conformational epitopes. Threeof the four anti S scFv clones recognised the RBDand retained an in vitro neutralising activity. Duanet al. also utilised a phage-display library techni-que to construct a
37、library from convalescentSARS patients. One of the scFv clones, B1, recog-nises the aa10231189 S2 protein region and haspotent neutralising activity 19.Greenough et al. obtained human mAbs againstSARS-CoVstructuralproteinsbyimmunisingtransgenic mice with human immunoglobulingenes 20,21. One of these
38、 mAbs, 201, recognisesthe RBD (aa490510) and another mAb, 68, recog-nises the aa130150 region, outside the RBD. Thus,a variety of approaches makes it possible to estab-lish mAbs of human origin against SARS-CoV. Ofinterest, the technique for establishing mAbs fromrecovered patients, in which high-af
39、finity antibo-dies with potent biological activity can be selectedduring virus elimination, is useful for other infec-tions to establish antibodies with potent activityfor diagnosis and, quite probably, for clinicalapproaches as well.Epitopes on SARS-CoV proteinsSpike (S) protein:SARS patients sera
40、recognisethe major conformational and linear S protein epi-topes 2224. The S protein of SARS-CoV (Gen-Bank accession no. 29836496) is 1225aa residueslong and contains a leader sequence at its N-term-inal 1,2 (Figure 1). The S protein is divided intotwo regions, S1 (aa1690) and S2 (aa6911255),althoug
41、h it is not cleaved by a protease as othercorona-viruses are. The S2 region contains a trans-membrane and cytoplasmic region and the S1 con-tainstheRBD.TheS1domainofGroup-Icoronaviruses, including human CoV-229E andtransmissible gastroenteritis virus (TGEV), recog-nises aminopeptidase N (CD13) as a
42、cellular targetmolecule 25,26. The binding site was mapped toaa407547 in the case of Hu-CoV 229E 27 andaa506655 in TGEV 28. Group-II coronaviruses,such as mouse hepatitis virus (MHV), recognisethe carcinoembryonic antigen-related cell adhe-sion molecule (CEACAM1) as a cellular receptor29, and the N-
43、terminal 330aa residues containFigure 1. Schematic drawing of S- and N-protein epitopes and the name of the recognizing mAbs. Small arrows indicate that the epitopesare confined to the peptide fragment indicated in the figure. Large arrows indicate that the epitopes are nearly confined to the region
44、indicated. The epitopes identified only in polyclonal antibodies are not listed120120Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.120120Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.Copyright # 2006 John Wiley & Sons, Ltd.Rev. Med. Virol. 2006; 16: 117131.Table 3. SARS Vaccine Studies i
45、n Animal ModelsType ofVaccineTarget orAnimalRoute andProtection Referencesvaccinespreparationantigenmodelimmunecontrol etcInactivatedSARS-CoVUV-irradiationWhole virionBALB/cs.c., twice,Nab and T-cellactivationND56Formaline-?-propyolactoneWhole virionWhole virionBALB/cBALB/cNabNabNDND65,6766Recombina
46、ntvirus vectorsAttenuatedvaccinia (MPA)SSBALB/cBALB/ci.n.i.m., twicei.m.6970(S-DNA)Rabbiti.m.NDMonkeyi.n versus i.m.SFerreti.p. or s.c.No effect71hepatitisAdenovirus type5S1, M, NMonkeyNab,N-peptide-reactive TND73NB6N-reactive TND90SB/6 andBALB/cS-peptide-reactive TND64epitopes in S1CD4 and CD8epito
47、pesParainfluenza virus SMonkeyNab77S, E, M, NHamsterNab, S only76RabiesvirusS and NBALB/ci.m., Nab, SonlyND74VSVSBALB/ci.n., Nab,passive imm.75DNA vaccinesS, SdTM,BALB/cNab, nocontribution of79SCIDCD4/CD8T cellsN-linked toB6CTL? (N80CalreticulinN peptidesVaccina)NBALB/cCTL:N-expressingcellsND81,82M,
48、 NB/6,CTL responseND91SCIDRecombinantproteinSolublepolypeptideS 14-762BALB/cNab, withadjuvant(MPLTDM)85Nab, neutralizing antibody; i.m, intramuscular; i.n, intra nasal; i.p., intraperitoneal; s.c., subcutaneous; ND, not done.Severe acute respiratory syndrome coronavirusSevere acute respiratory syndr
49、ome coronavirus121121Severe acute respiratory syndrome coronavirusSevere acute respiratory syndrome coronavirus121121Copyright # 2006 John Wiley & Sons, Ltd.Rev. Med. Virol. 2006; 16: 117131.the receptor binding site 30. Upon binding to thetarget molecule, the S protein undergoes a confor-mational c
50、hange leading to exposure of the fuso-genicregionintheS2partresultinginmembrane fusion between the viral and cellularmembranes 31,32.Mouse mAbs against recombinant protein frag-ments of S-II (aa485625), S34 and S78, obtained byZhou et al. 13, had potent neutralisation ability.The epitopes of S34 and
51、 S78 were localised toaa548567 and aa607627, respectively. These epi-topes, however, are slightly downstream of theRBD shown by Wong et al. 9 and Xiao et al.33. On the other hand, among the mAbs againstRBD fused with human IgG1-Fc fragment (RBD-Fc), two mAbs, 4D5 and 17H9 recognised linearepitopes s
52、uch as aa435451 and aa442465, respec-tively 34, whereas the remaining mAbs recog-nized conformational epitopes, classified into sixgroups as Conf-I to Conf-VI. This study showedthat the ability to inhibit ACE2 binding of SARSpseudovirus was retained in all conformationalepitopes, especially in Conf-
53、III, -IV and V, butnot in the linear epitopes.Likewise,humanmAbsagainstSprotein,such as 80R 35, CR3014 36 and 201 recognisethe RBD and have potent neutralising activityagainst SARS-CoV, as discussed below. The clone,CR3014, recognises the S1 fragment in the contextwith aa479, suggesting the importan
54、ce of this ami-no acid in the high binding affinity of CR3014.Spiga et al. tried to model the tertiary structureof the S1 and S2 S protein domains by the homol-ogy modelling and molecular dynamics methods37. Their model predicted two hydrophobicpockets, Phe850-Phe870 and Phe10771079, forthe putative
55、 receptor binding site. This modelwould be useful in evaluating the antigen driftcaused by the mutations in the S1 region, whichhas already been deposited in the NCBI database.Interestingly, when Yi et al. studied the immuno-genicity of S protein in mice by DNA immuniza-tion, a single amino acid sub
56、stitution, the R441Amutation, failed to induce neutralizing antibodiesand abolished viral entry. The R453A mutation,however, retained the capacity to induce neutraliz-ing antibodies, although it also abolished viralentry 38. Thus, a single amino acid mutationhere easily affects the virus-to-cell int
57、eraction,and therefore, this region is an ideal target struc-ture for neutralization.Nucleocapsid (N) protein:The nucleocapsid (N)protein of SARS-CoV is a highly basic structuralprotein of 422 amino acids. The N protein isthought to be involved in the packaging of theviral RNA-genome and, thus, cruc
58、ial for viralreplication and pathogenesis as in other CoVs39,40 (Figure 1). Anti-N-protein antibodies aregenerally found in the sera of SARS patients, sug-gesting that the N-protein is one of the immuno-dominantstructuralproteinsofSARS-CoV41,42. In addition, SARS patients sera recognisethe N-protein
59、 at a large number of linear epitopes23,24and, at least, two major conformationalepitopes 43.In patients sera, Wang et al. identified the pep-tides N66 (aa6687) and N371401 (aa371422)as the most immunogenic epitopes 24. Onthe other hand, He et al. identified two major epi-topes in the C-terminal reg
60、ion (aa362412) andmiddle region (aa153178), in addition to severalminor immuno-dominant epitopes 44. They alsoshowed that the most potent antibody responsewas elicited against the C-terminal region, whichcontains a short lysine-rich sequence (aa362381;KTFPPTEPKKDKKKKTDEAQ); however, the func-tional
61、role of this stretch remains unknown. Vanden Brink et al. established two human anti-N scFvs, CR3018 and CR3009, which recogniseda linear epitope, RSAPRITFG (aa1119) and anonlinear epitope, respectively 18.In mice immunised with inactivated SARS-CoV,we identified one major epitope in the middlepart
62、(aa110210), which was recognised both bythe SKOT-8 and SKOT-9mAb 10, whereas Heet al. identified one major epitope adjacent tothe N-terminal region (aa76101) in addition toepitopes in the C-terminal and middle regions44.Together, these mapping studies of the SARSCoV N protein suggest that there are
63、three majorepitopeslocalisedtotheN-terminal,middleand C-terminal regions, respectively, which areresponsible for the potent immune response inboth humans and mice.Envelop (E) protein:The small E protein is a 76aaresidue long protein involved in the envelop mor-phogenesis. As far as we know, there is
64、 only onemAb against this protein in humans 16, andthis E protein antibody is detected in SARS conva-lescent patient sera 23.122122Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.122122Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.Copyright # 2006 John Wiley & Sons, Ltd.Rev. Med. Virol. 20
65、06; 16: 117131.Matrix (M) protein:The M membrane glycopro-tein is a 221aa residue long integral membraneprotein which is functionally involved in the bud-ding of virions from cells. Pang et al. reported thatrabbit antiserum raised against recombinant Mprotein has a potent neutralising ability in vit
66、ro,suggesting that the M protein could be one of thetarget proteins chosen for the vaccine develop-ment 45. Wang et al. reported that the syntheticpeptide M137 (aa137158) is one of the mostimmunogenic regions of the SARS-CoV structuralproteins 24. Recently, Zhong et al. reported a sys-tematic search
67、 for the viral epitopes by a bio-panning of the M13 phage display dodecapeptidelibrary using antibodies found in plasma samplesof convalescent SARS patients 46. They determinedthe continuous viral epitopes including an epitope-rich region on the S2, M and E proteins. This contig-uous epitope map of
68、SARS-CoV would greatly helpto develop an effective vaccine for SARS-CoV.Antibody cross-reactivity to OC43 and229EWe confirmed the absence of cross-reactivity in theanti-N mAbs, SKOT-8 and SKOT-9, to humancoronaviruses, HCoV-OC43 and HCoV-229E, byELISA 10 and IFA (Figure 2). However, at thepolyclonal
69、 level, there are reports of cross-reactiv-ity between SARS and other human coronaviruses.Wo et al. reported false-positive results in theirrecombinant SARS-CoV N-protein-based ELISAassay, mainly due to the presence of cross-reactiveFigure 2. Immuno-fluorescent assay showing the absence of cross-rea
70、ctivity with anti-SARS-CoV S-protein mAb, SKOT-3 and anti-N-protein mAb SKOT8, with Hu-CoV 229E and OC43. SARS-CoV-infected VeroE6 cells (upper lane), Hu-CoV 229E-infected L132 cells(middle lane) and Hu-CoV OC43-infected HRT-18 cells are stained with anti-SARS-CoV S- and N-protein mAbs. SKOT-3 (left
71、 columns)and SKOT-8 (middle column) and polyclonal anti-BCV/CCV (right columns)Severe acute respiratory syndrome coronavirusSevere acute respiratory syndrome coronavirus123123Severe acute respiratory syndrome coronavirusSevere acute respiratory syndrome coronavirus123123Copyright # 2006 John Wiley &
72、 Sons, Ltd.Rev. Med. Virol. 2006; 16: 117131.antibodies to SARS-CoV N-protein in the sera 47.They found that 3 out of 21 and 1 out of 7 serafromconvalescentpatientsfromHCoV-OC43and HCoV-229E, respectively, contained antibo-dies cross-reactive with the SARS-CoV N-protein,suggesting the presence of a
73、common epitope(s)in these human CoV N-proteins. The sera fromconvalescent patients, however, did not reactwith SARS-CoV S-proteins in the Western blotanalysis. Thus, highly specific SARS-CoV mAbsare required for sensitive and accurate laboratorydiagnosis.NEUTRALISING ABILITYOF ANTI-SARS-CoV ANTIBODI
74、ESNeutralising antibodies are crucial for establishingprotection from SARS-CoV infection. Notably, theanalysis of the memory B cell repertoire at a clonallevel suggested that the memory B cell repertoirein recovered SARS patients is biased towards neu-tralising antibodies recognising the S protein 4
75、8.As described above, the main neutralising S pro-tein epitope was at the aa318510 position, whichbinds to the cellular receptor ACE2 (RBD; seeFigure 1). Therefore, under the expectation that apassive immunotherapy that utilises neutralisingantibodies against SARS-CoV could be a promis-ing therapeut
76、ical method, many efforts havebeen focused on the development of mAbs as dis-cussed above.Analysis of rabbit anti-sera against recombinantS, N and M proteins suggested significant neutra-lising abilities in anti-S and anti-M antibodies, butnot in anti-N antibodies 45. The main neutralis-ing epitope
77、on S protein was in the RBD, whereassome parts irrelevant to the ACE2 binding sitewere also shown to be involved in neutralisation,presumably via steric hindrance. Furthermore, acompetitive neutralisation assay suggested thepresence of an additional target site other thanACE2 binding site 45. In thi
78、s regard, by continu-ous epitope mapping using the M13 phage displaydodecapeptide library, Zhong et al. reported thattheepitope-richregionontheS2protein(aa787809) represents one of the major neutralis-ing targets for SARS patients and that is blockedby the peptide of the corresponding region 46.Huma
79、n mAbs against SARS-CoV with neutra-lisingactivityareattractivereagentsforthetreatment of SARS patients. For example, allEBV-transformed memory B cells recovered fromSARS patients have specificity for the S proteinwith neutralising activity 48. Curiously, one ofthem, S3.1, conferred efficient protec
80、tion in amouse model with SARS-CoV infection 16. Inaddition, one of mAbs established from the humanscFv library by Sui et al., designated 80R, wasshown in a mouse model to display effectiveprotection from infection with wild-type or mutantviruses isolated from patients 49. Furthermore,when80RIgG1was
81、administeredintothemice prophylactically, SARS-CoV replication wasreduced by more than four orders of magnitudeas compared with untreated mice. Human mAbs,201, established from transgenic mice that har-boured human immunoglobulin genes 20 alsohad neutralising activity and significant protectiveeffec
82、t in the mouse infection model upon adminis-tration with a 1.640mg/kg dose 21. Together, allthese human mAbs could be candidates for che-moprophylaxis and therapy for SARS, and, infact, clinical trials are being planned for some ofthese human mAbs 16,18,21,49.APPLICATION OF ANTI-SARS-CoVmAbs FOR LAB
83、ORATORY DIAGNOSISLaboratory diagnosis of SARS-CoV infection wasbased on a combination of serologic tests, reversetranscription-polymerase chain reaction (RT-PCR)and virus isolation 5053. Hence, the mAbsagainst SARS-CoV structural proteins have beenestablished by several groups and their utility inan
84、tigen-captureELISA,immuno-fluorescenceassay, Western blot analysis has been tested10,51,54(see Table 1).Immuno-fluorescence assay (IFA)and immuno-histochemistry (IHC)IFA is the simplest serological test for detectingthe pathogen. The IFA for SARS diagnosis is car-ried out by using several polyclonal
85、 and mono-clonalantibodiesagainstN-proteinorthoseagainst S-protein, irrespective of the specificityforconformationalorlinearepitopes10,15,18,53. We showed that SKOT-8 and SKOT-9mAbs against N protein were quite useful fordiagnosis,intermsofbothspecificityandsensitivity 10.He et al. developed a syste
86、m that uses insectsf9 cells expressing the N195-Sc fusion protein asa target cell, by which 23 serum samples fromSARS patients were scored at a rate comparable124124Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.124124Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.Copyright # 2006 John Wil
87、ey & Sons, Ltd.Rev. Med. Virol. 2006; 16: 117131.to that of conventional IFA and a commercialSARS-CoV IFA kit 55.Western blot:The majority of anti-N mAbs aredirected against linear epitope(s) and thus usedin Western blot to detect the 47kDa band of theN-protein 10,15. In contrast, the epitopes onnat
88、ive S-protein are mostly conformational andthe anti-S mAbs we obtained were not useful forWesternblot10.However,byusingSDS-denatured S protein or recombinant S protein frag-ments as an immunogen, several mAbs for Wes-tern blot were obtained 11,13. Although thesera from patients and from immunised mi
89、cedetected both the E and M protein 23,24,56, nouseful mAb for detecting either of these proteinsby Western blot have been established.ELISA:Because the immunological test for thepresence of SARS-CoV virions in a patients speci-men is an important complement to the RT-PCRtest, three groups have so f
90、ar reported the devel-opment of such an antigen capture ELISA systemfor SARS-CoV. We intended to establish an anti-gen-capture sandwich ELISA system by using atotal of 29 mouse mAbs with specificity for S-and N-proteins, and found that a virus proteinload as low as 40pg/mL was successfully detectedb
91、y use of the anti-N mAb, SKOT-8, as a capturingantibody and the biotinylated mAb, SKOT-9, as aprobing antibody 10. The sensitivity of the sys-tem did not differ between at least four strains ofSARS-CoV tested. In contrast, any combination ofanti-S mAbs failed to yield high titres.Che et al. 15 utili
92、sed three mouse anti-N mAbsas a capturing antibody and a rabbit polyclonalanti-N antibody as a detecting antibody, andwere able to detect the recombinant N protein at50pg/mL. This assay system scored positive forapproximately 84% of SARS patients serologicallyconfirmed and 1.5% in 1272 healthy indiv
93、iduals15.Di et al. tested 829 serum samples from 643patients and compared them to 197 control serafrom healthy donors 57. After the onset of symp-toms, the N protein was detected by day 10 with asensitivity of 100% in the case of 27 patients whohad been positive with the neutralisation test.After 10
94、 days, the N-protein level in the seradecreased and was undetectable beyond days 19.The specificity of the assay calculated from theresults of 66 serological test-negative patients and197 healthy donors was reported to be 100%. Thepositive rate, 96100%, was obtained from the seraat day 35 after the
95、onset of symptoms for 27 neu-tralisation-test positive SARS patients and 298 ser-ologically confirmed patients.Lau et al. reported the development of polyclo-nal antibody-based ELISA for the N protein by uti-lising guinea pig and rabbit antibodies againstrecombinantHis6-taggedSARS-CoVN-protein58. Th
96、ey tested nasopharyngeal aspirates, urineand faecal samples, and detected N protein inthe nasopharyngeal aspirate samples (day 624after the onset of the disease), in urine samples(day 1131) and in faecal samples (day 832),though the sensitivity was not very high exceptfor the day 1115 nasopharyngeal
97、 aspirates (83%sensitivity).Apart from an antigen-capture detection sys-tem, the high-throughput assay for anti-SARS-CoV IgG antibody detection system, DETECT-SARSTM, was reported to be highly sensitive(95.9%) for convalescent serum samples 59.These immunological detection systems wouldbe necessary
98、compliments of RT-PCR-based diag-nosis, and any future improvement in these detec-tion systems will certainly contribute to control theSARS pandemic.IMMUNITY TO SARS-CoVAND ANIMAL MODELS (Table 3)Immune responses have been studied using ani-mal models including macaques, ferrets, cats,Golden Syrian
99、hamsters and mice (review in 3).However, these animal models showed only tran-sient viral replication in the respiratory tracts with-out manifestation of the disease, except in ferretsandhamsters.Ferretsandhamstersdeveloppathological disorders in the lung and infected fer-rets and cats transmit SARS
100、-CoV to na ve counter-parts housed together 60. Thus, it seems thatsomeaspectsoftheSARS-CoVinfectioninhumans were reproduced in these animal models,but the disease entities themselves are quite differ-ent. For example, in humans, a rapid progressionof pulmonary and intestinal complications isobserve
101、d and almost 20% of SARS patients devel-op watery diarrhoea 61, which is not seen in ani-malmodels.Despitethedifferentoutcomes,animal models are still useful for vaccine develop-ment and evaluation.Severe acute respiratory syndrome coronavirusSevere acute respiratory syndrome coronavirus125125Severe
102、 acute respiratory syndrome coronavirusSevere acute respiratory syndrome coronavirus125125Copyright # 2006 John Wiley & Sons, Ltd.Rev. Med. Virol. 2006; 16: 117131.While antibody responses in SARS patients havebeen studied extensively, we know little aboutT-cell responses in patients. Two A-2-restri
103、ctedepitopes of the S2 domain were recognised bymemoryCD8TcellsofSARS-CoV-infectedpatients 62. However, a study using HLA-A0201 transgenic mice or in vitro primed PBMCsof healthy donors identified a slightly differentdominant epitope in the S2 domain 63. In mice,epitopesof CD8T cell were within the
104、S1domain 64. Current strategies to elicit an effectiveimmunity against SARS-CoV are listed in Table 3.VACCINE STUDIESAttenuated or inactivated SARS-CoVBecause we know little about the pathogenesis ofsevereacuterespiratorysyndromeassociatedwithSARS-CoVinfection,aliveattenuatedSARS-CoV vaccine will no
105、t be feasible as a candi-date for a SARS vaccine. Instead, a whole inacti-vatedvirionwillbethefirstchoice.Micesubcutaneously or nasally injected with a wholevirion, that had been inactivated either by UV-irradiation 56 or formalin 6567, with or with-out a variety of adjuvant, induced a high level of
106、antibodies against SARS-CoV. Surprisingly, weobserved that the UV-inactivated virion per se eli-cited a considerably high level of serum IgG-typeneutralising antibody without an alum adjuvant56. Furthermore, the level of serum IgG antibodywas retained at the peak for more than 6 monthsafter a single
107、 injection, probably reflecting the gen-eration and maintenance of long-term AFCs (anti-body-forming cells, i.e. effector memory cells) 68.No IgA response was elicited in mucosal tissues bysubcutaneous injection, whereas a response waselicited in mice immunised nasally with inacti-vated virions by t
108、he aid of potent mucosal adju-vants, such as cholera toxin (CT) or CpG 66.TheChinesegovernmentrecentlyannouncedthat they completed a phase I study of an inacti-vated virion vaccine, and that no serious complica-tions were observed so far (http:/ vaccinesVirus vector-based vaccines:As described in th
109、eprevious section, S and N proteins are two majorviral proteins which induce a high level of anti-body response. Current strategies to develop aneffective vaccine generally rely on the inductionof potent neutralising antibodies on the mucosalsurfaces. The study of attenuated parainfluenzavirus sugge
110、st that immunisation with recombinantS proteins, but not E, M or N proteins, results inprotection by eliciting neutralising antibodies,indicating the advantage of S antigen-expressingvaccines currently in development.Likewise, the modified vaccinia virus Ankara(MVA) expressing the S protein (MVA-S)
111、hasbeen used successfully in mice 69, rabbits andmonkeys 70. MVA-S vaccination induced a highlevel of neutralising antibodies, which in turnresults in protecting the monkeys from virus infec-tion. In contrast, inoculation of attenuated vacciniaexpressing the S protein into ferrets had no effecton th
112、e viral load and caused hepatitis uponchallenge with SARS-CoV in MVA-S-immunisedferrets71.However,itremainsunknownwhether this has been caused by the mechanismofantibody-dependentenhancementofvirusinfectivity or some other immunopathologicaleffect on the liver. In feline coronavirus infection,an ant
113、ibody-dependent enhancement of the dis-ease was observed in vaccinated animals withvaccinia vector 72. Thus, such an unfavourableside effect of MVA-S vaccination has to be care-fully considered for application.Adenovirus type 5 vector expressing codon-optimised S, M or N was also developed as oneof
114、the candidate vaccines 73. When six monkeyswere immunised with these three recombinantadenoviruses, all developed antibodies against S1fragments with neutralising activity. They alsodeveloped various levels of IFN-?-producing Tcells reactive to pooled 15-mer N peptides. How-ever, no challenge experi
115、ment was carried out.Whether or not pre-existing immunity againstadenovirus reduces the efficacy of recombinantadenovirus vaccine in humans needs to be furtherinvestigated.Rhabdovirus-based vaccines were also devel-oped, using a recombinant technology for rabiesvirus (RV) 74 and vesicular stomatitis
116、 virus(VSV) 75. Both viruses, which encode the S pro-tein in their genome, induced a high level of neu-tralising antibodies after a single injection intomice. In the case of VSV-based vector, pre-existingimmunity is not a concern, and mice were pro-tected from SARS-CoV infection by intranasalimmunis
117、ation. RV can replicate in mucosal mem-brane cells and this is one of the advantages of126126Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.126126Y. Tsunetsugu-YokotaY. Tsunetsugu-Yokotaet al.et al.Copyright # 2006 John Wiley & Sons, Ltd.Rev. Med. Virol. 2006; 16: 117131.an RV-based vaccine. In
118、tramuscular injection ofRV-based vector expressing N protein did not eli-cit a neutralising antibody. Of note, the size of theS protein expressed by recombinant RV is smaller(?140kDa) than that observed in other reports(?200kDa) 10,69,7577, suggesting that the Sprotein may not be modified by glycosy
119、lation dur-ing the replication of RV. In any case, the protec-tive ability of the RV-based vaccine needs to betested further.DNA vaccine:DNA immunisation has been devel-oped as a safe and stable vaccine technology forprotection against a variety of infectious diseases.Since then, many efforts have b
120、een made toimprove the efficiency of DNA vaccine, for exam-ple, by adding cytokines and costimulatory DNAadjuvants (review in 78). Using modified codonsto optimise expression, Yang et al. prepared plas-mids encoding S protein and analysed their abilityto elicit antiviral immunity in mice 79. BothCD4
121、and CD8T cells reactive to overlapping Speptides were detected and these mice generatedneutralising antibodies, which accounts for theirprotection from respiratory infection. Depletionof both CD4and CD8T cells or adoptive T-cell transfer of immunised T cells did not affectprotective immunity, wherea
122、s passive transfer ofIgG from immunised mice provided immune pro-tection. This study, along with that of attenuatedparainfluenza virus 76, indicated that humoralimmunity against S protein alone can confer pro-tection. However, since SARS-CoV replicates inmice only transiently, it remains to be eluci
123、datedwhether T-cell immunity is also important espe-cially when neutralising antibodies fail to clearthe virus.The other studies used the plasmid-expressingN protein, expecting that a high level of intracellu-lar N protein expression may induce strong CTLresponses 8082. In order to increase the effi
124、-ciency, Kim et al. 80 linked the calreticulin (CRT)gene to the N gene, which is a Ca2-binding, heatshock family protein abundantly present in theendoplasmic reticulum. When mice were injectedthree times with CRT/N DNA by a gene gun,humoralandT-cellimmuneresponsewereenhancedandtheseimmunisedmicewere
125、protected from infection by recombinant vacciniaexpressing N protein. This is the only approachso far which demonstrated the protective role ofN-reactive T cells by vaccination. However, itremains unknown whether or not N-reactive Tcells can contribute to suppressing the progressionof SARS during na
126、tural infection.Lastly, a combination of DNA vaccine withrecombinant S protein 83 or whole-killed virus84 were designed, with the expectation that thisregimen may enhance not only the titre of neutra-lising antibody but also T-cell response, thoughthe importance of the latter for protection is notye
127、t clear.Recombinant proteins for vaccination:Because theS protein in SARS-CoV is as heavily glycosylatedas is the gp120 of HIV, the large scale productionof recombinant protein is economically difficult.Nevertheless, a component vaccine has a greatadvantage with respect to safety, if it is effective
128、enough. A baculovirus system was developed byBisht et al. and they produced soluble recombinantpolypeptide containing an N-terminal segment ofthe spike glycoprotein (nS) 85. When mice wereinjected subcutaneously three times with nS witha Ribi (MPLTDM) adjuvant, they were protectedfrom SARS-CoV infec
129、tion 4 weeks later. It wouldbe interesting to study whether the neutralisingantibody elicited by this protein subunit vaccineis sustained and is able to protect susceptible ani-mals from disease.CONCLUSIONThe mAbs against the structural proteins of SARS-CoV have been rapidly established and are bein
130、gapplied not only for basic research but also in clin-ical practice, including SARS patient therapy. Stu-diesutilisingtheseantibodiesprovideusenormous information with respect to the immuneresponses elicited by the SARS-CoV and for thedevelopment of vaccines against the SARS-CoV.What we learned from
131、 the emergence of SARSwill be potently useful for preparing against anyfuture outbreaks of new pathogens likely comingfrom wild animals.ACKNOWLEDGMENTWe thank a member of the Department of Immu-nology, who shared this work regarding mAbestablishment and basic SARS-Co vaccine devel-opment. We are als
132、o grateful to Mr. HirotakaTakagi for preparing cell lines infected withhuman coronaviruses. This work was supportedSevere acute respiratory syndrome coronavirusSevere acute respiratory syndrome coronavirus127127Severe acute respiratory syndrome coronavirusSevere acute respiratory syndrome coronaviru
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