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Rev Med Virol 2006 16 117 131 Published online in Wiley InterScience Reviews in Medical Virology DOI 10 1002 rmv 492 Severe acute respiratory syndrome SARS coronavirus application of monoclonal antibodies and development of an effective vaccine Yasuko Tsunetsugu Yokota Kazuo Ohnishi and Toshitada Takemori Department of Immunology National Institute of Infectious Diseases Toyama cho Shinjuku ku Tokyo Japan SUMMARY SARS 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 monoclonal antibodies mAbs against SARS CoV have been developed and applied for diagnosis clinical management and basic research In this review we overview the biochemical and functional properties and applications of these SARS CoV mAbs We also focus on a variety of vaccines currently under development and 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 Revised 13 December 2005 Accepted 13 December 2005 INTRODUCTION The outbreak of fatal severe acute respiratory syn drome SARS which originally occurred in 2002 in China and subsequently spread to many countries in early 2003 is now under control due to a con certed world wide effort led by the World Health Organisation WHO A causative pathogen SARS CoV was identified and the entire genome sequence was quickly determined 1 2 SARS CoV belongs to a distinct group of known human coronaviruses such as HCoV 229E Group 1 and HCoV OC43 Group 2 which are only slightly pathogenic in humans On the contrary animal coronaviruses have been known to cause clinically serious diseases in livestock and pet animals Based on the nucleotide sequence homology of SARS CoV like virus in wild animals traded in Chinese markets it is considered that SARS CoV emerged through the interspecies transmission of such animal coronaviruses review in 3 A recent report strongly suggested that the Chinese horseshoe bat was a natural host of SARS CoV bat SARS CoV and that caged animals such as Himalayan palm civets and raccoon dogs are the amplification hosts 4 SARS CoV is readily transmissible through close contact between family members and health care and laboratory workers Since SARS CoV infects to and replicates in the respiratory and intestinal tissues a vaccine eliciting a mucosal neutralising antibody particularly in the broncho alveolar lumen would be most desirable It has been reported that high titres of neutralising IgG antibo dies against SARS CoV are present in SARS patient 5 and that passive administration of ser um antibodies from previously infected patients improved the conditions in newly infected recipi ents 6 Furthermore in the mouse model the passive transfer of mouse immune serum against SARS CoV has been demonstrated to reduce pulmonary viral titres in mice infected with SARS CoV 7 These results support the notion that the neutralising antibodies are crucial for protection against SARS CoV infection On the occasion of the SARS outbreak several laboratories endeavoured to identify the mono clonal antibodies mAbs that act against SARS CoV in order to provide tools for diagnosis in R E V I E W Copyright 2006 John Wiley 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 been established and are listed in Table 1 for mouse and Table 2 for human mAbs In addition the fine epitope mapping studies of these mAbs are summarised in Figure 1 We now know that the S protein binds to the cellular receptor ACE2 8 and that ACE2 recognises the S protein at the ami no acid residues aa318 510 9 This region is designated as a receptor binding domain RBD Thus the S protein especially RBD is a major target for eliciting neutralising antibody We will focus 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 of SARS CoV as an immunising antigen which had been inactivated by UV irradiation to avoid unwanted denaturation of virus protein 10 The majority of mAbs established 26 out of 29 recog nised conformational epitopes on the S protein with virus neutralising ability in vitro whereas 3mAbs recognised linear epitopes on the N pro tein On the other hand Chou et al established several mAbs which recognise the linear epitopes of S protein by using detergent disrupted virions as an antigen 11 Such mAbs designated 1A5 and 2C5 had high neutralisation ability suggest ing that neutralising activity is mediated by a vari ety of antibody recognition sites on the S protein Gubbins et al used whole virions inactivated with C12 propiolactone and obtained 9mAbs with virus neutralising ability 12 Surprisingly the majority of mAbs are encoded by the V H gene family V H J558 coupled with variety of light chains sug gesting a correlation between neutralisation ability and specific V H gene allele Table 1 Establishment of mouse monoclonal antibodies Host Antigen adjuvant Clones Application Reference 1 Whole virion 26 clones of anti S and Sandwich ELISA Western blot 10 UV inactivated FCA 3 clones of anti N Histology IFA neutralization SKOT 8 C09 etc 2 Whole virion 7 clones of anti S Epitope mapping on S 86 detergent inactivated 1A5 2C5 etc Neutralization FCA Western blot 3 Whole virion 5 clones to S MAbs belong to 87 beta propiolactone F26G18 F26G19 etc single V H gene family inactivated FCA 4 Recombinant N 9 clones to anti rN Sandwich ELISA Western blot 88 MPL TDM a NE4A4 NE8A11 etc 5 Recombinant S 4 clones to S Neutralization Western blot 13 fragment FCA S26 S34 S84 S78 ELISA cyto staining 6 RBP Fc b 27 RBP specific mAbs Epitope mapping 14 4D5 17H9 etc Neutralization a MPL monophosphoryl lipid A TDM trehalose dicorynomycolate b RBP Fc fusion protein containing the receptor binding domain RBP linked to a human IgG1 Fc fragment 118 Y Tsunetsugu Yokota et al Copyright 2006 John Wiley 16 117 131 Another approach is to use the recombinant SARS CoV proteins as an immunising antigen Zhou et al prepared a recombinant protein frag ment of S protein S II aa 485 625 which was pre dicted to include the RBD 13 This recombinant protein fragment binds to the surface of Vero cells and the mAbs against this fragment had potent in vitro neutralisation ability He et al used a small fragment of recombinant RBD which was fused with human IgG1 Fc fragment RBD Fc as an immunising antigen 14 Twenty seven hybrido mas were established and their antigen specifici ties were mapped into 6 different conformation dependent and 2 adjacent linear epitopes see below 14 Using recombinant N protein Che et al isolated anti N mAbs useful for the sensitive antigen capture ELISA system and Western blot 15 Human mAbs Table 2 Considering the clinical approach to block SARS CoV infection by utilising neutralising antibodies human mAbs have been established by different techniques Traggiai et al developed a unique method of EBV transformation of B cells which enabled rapid generation of human neutralising antibodies against SARS CoV 16 The memory B cell pools of recovered SARS patients were effi ciently immortalised and high affinity 10 C08 Mto 10 C011 M human mAbs were successfully obtained In this case about 80 of EBV transformed B cell clones secreted IgG antibody against S protein and about 15 against N protein most likely reflecting the actual memory B cell repertoire in those individuals who recovered from SARS CoV infection Sui et al obtained eight clones of two single chain variable antibody fragments scFvs by screening the scFv library constructed from non immune human antibodies on the S1 fragment of SARS CoV S protein 17 Among these clones 80R bound to the S protein RBD region and showed an efficient neutralisation ability 17 Using a similar approach van den Brink et al Table 2 Human monoclonal antibodies for SARS CoV Method Clones Notes References Improved EBV transformation of memory B cells from patients anti S N E many clones S3 1 S102 1 etc In vitro neutralization activity 10 C08 10 C011 M In vivo protection in mouse model 48 scFv a phage display screening on recombinant S1 protein 8 scFv clones 80R etc Memory B cell repertoire representation Epitope mapping in vitro neutralization 35 In vivo protection in mouse model 89 S1 genotyping co related with 80R sensitivity scFv phage display screening on irradiated SARS CoV whole virion anti S N CR3014 CR3018 CR3009 Epitope mapping immunoelectron microscopy IFA in vitro neutralization 36 scFv the library constructed from SARS convalescent patients selection against inactivated whole virion anti S2 B1 etc Mapped to 1023 1189 of S2 protein potent neutralization activity in vitro 19 Transgenic mice with human immunoglobulin gene Medarex immunized with recombinant S protein 2 clones 68 201 Epitope mapping In vivo protection in mouse model 21 a scFv single chain variable antibody fragments Severe acute respiratory syndrome coronavirus 119 Copyright 2006 John Wiley 16 117 131 obtained eight scFv clones of which two clones could be mapped to the N protein and four to the S protein 18 One of the two anti N scFv clones recognises a linear epitope and all of the others recognise conformational epitopes Three of the four anti S scFv clones recognised the RBD and retained an in vitro neutralising activity Duan et al also utilised a phage display library techni que to construct a library from convalescent SARS patients One of the scFv clones B1 recog nises the aa1023 1189 S2 protein region and has potent neutralising activity 19 Greenough et al obtained human mAbs against SARS CoV structural proteins by immunising transgenic mice with human immunoglobulin genes 20 21 One of these mAbs 201 recognises the RBD aa490 510 and another mAb 68 recog nises the aa130 150 region outside the RBD Thus a variety of approaches makes it possible to estab lish mAbs of human origin against SARS CoV Of interest the technique for establishing mAbs from recovered patients in which high affinity antibo dies with potent biological activity can be selected during virus elimination is useful for other infec tions to establish antibodies with potent activity for diagnosis and quite probably for clinical approaches as well Epitopes on SARS CoV proteins Spike S protein SARS patient s sera recognise the major conformational and linear S protein epi topes 22 24 The S protein of SARS CoV Gen Bank accession no 29836496 is 1225aa residues long and contains a leader sequence at its N term inal 1 2 Figure 1 The S protein is divided into two regions S1 aa1 690 and S2 aa691 1255 although it is not cleaved by a protease as other corona viruses are The S2 region contains a trans membrane and cytoplasmic region and the S1 con tains the RBD The S1 domain of Group I coronaviruses including human CoV 229E and transmissible gastroenteritis virus TGEV recog nises aminopeptidase N CD13 as a cellular target molecule 25 26 The binding site was mapped to aa407 547 in the case of Hu CoV 229E 27 and aa506 655 in TGEV 28 Group II coronaviruses such as mouse hepatitis virus MHV recognise the carcinoembryonic antigen related cell adhe sion molecule CEACAM1 as a cellular receptor 29 and the N terminal 330aa residues contain Figure 1 Schematic drawing of S and N protein epitopes and the name of the recognizing mAbs Small arrows indicate that the epitopes are confined to the peptide fragment indicated in the figure Large arrows indicate that the epitopes are nearly confined to the region indicated The epitopes identified only in polyclonal antibodies are not listed 120 Y Tsunetsugu Yokota et al Copyright 2006 John Wiley 16 117 131 Table 3 SARS Vaccine Studies in Animal Models Type of Vaccine Target or Animal Route and Protection References vaccines preparation antigen model immune control etc Inactivated SARS CoV UV irradiation Whole virion BALB c s c twice Nab and T cell activation ND 56 Formaline C12 propyolactone Whole virion Whole virion BALB c BALB c Nab Nab ND ND 65 67 66 Recombinant virus vectors Attenuated vaccinia MPA S S BALB c BALB c i n i m twice i m 69 70 S DNA Rabbit i m ND Monkey i n versus i m S Ferret i p or s c No effect 71 hepatitis Adenovirus type5 S1 M N Monkey Nab N peptide reactive T ND 73 N B6 N reactive T ND 90 S B 6 and BALB c S peptide reactive T ND 64 epitopes in S1 CD4 and CD8 epitopes Parainfluenza virus S Monkey Nab 77 S E M N Hamster Nab S only 76 Rabiesvirus S and N BALB c i m Nab S only ND 74 VSV S BALB c i n Nab passive imm 75 DNA vaccines S SdTM BALB c Nab no contribution of 79 SCID CD4 CD8 T cells N linked to B6 CTL N 80 Calreticulin N peptides Vaccina N BALB c CTL N expressing cells ND 81 82 M N B 6 CTL response ND 91 SCID Recombinant protein Soluble polypeptide S 14 762 BALB c Nab with adjuvant MPL TDM 85 Nab neutralizing antibody i m intramuscular i n intra nasal i p intraperitoneal s c subcutaneous ND not done Severe acute respiratory syndrome coronavirus 121 Copyright 2006 John Wiley 16 117 131 the receptor binding site 30 Upon binding to the target molecule the S protein undergoes a confor mational change leading to exposure of the fuso genic region in the S2 part resulting in membrane fusion between the viral and cellular membranes 31 32 Mouse mAbs against recombinant protein frag ments of S II aa485 625 S34 and S78 obtained by Zhou et al 13 had potent neutralisation ability The epitopes of S34 and S78 were localised to aa548 567 and aa607 627 respectively These epi topes however are slightly downstream of the RBD shown by Wong et al 9 and Xiao et al 33 On the other hand among the mAbs against RBD fused with human IgG1 Fc fragment RBD Fc two mAbs 4D5 and 17H9 recognised linear epitopes such as aa435 451 and aa442 465 respec tively 34 whereas the remaining mAbs recog nized conformational epitopes classified into six groups as Conf I to Conf VI This study showed that the ability to inhibit ACE2 binding of SARS pseudovirus was retained in all conformational epitopes especially in Conf III IV and V but not in the linear epitopes Likewise human mAbs against S protein such as 80R 35 CR3014 36 and 201 recognise the RBD and have potent neutralising activity against SARS CoV as discussed below The clone CR3014 recognises the S1 fragment in the context with aa479 suggesting the importance of this ami no acid in the high binding affinity of CR3014 Spiga et al tried to model the tertiary structure of the S1 and S2 S protein domains by the homol ogy modelling and molecular dynamics methods 37 Their model predicted two hydrophobic pockets Phe850 Phe870 and Phe1077 1079 for the putative receptor binding site This model would be useful in evaluating the antigen drift caused by the mutations in the S1 region which has 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 substitution the R441A mutation failed to induce neutralizing antibodies and abolished viral entry The R453A mutation however retained the capacity to induce neutraliz ing antibodies although it also abolished viral entry 38 Thus a single amino acid mutation here easily affects the virus to cell interaction 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 structural protein of 422 amino acids The N protein is thought to be involved in the packaging of the viral RNA genome and thus crucial for viral replication and pathogenesis as in other CoVs 39 40 Figure 1 Anti N protein antibodies are generally found in the sera of SARS patients sug gesting that the N protein is one of the immuno dominant structural proteins of SARS CoV 41 42 In addition SARS patient s sera recognise the N protein at a large number of linear epitopes 23 24 and at least two major conformational epitopes 43 In patient s sera Wang et al identified the pep tides N66 aa66 87 and N371 401 aa371 422 as the most immunogenic epitopes 24 On the other hand He et al identified two major epi topes in the C terminal region aa362 412 and middle region aa153 178 in addition to several minor immuno dominant epitopes 44 They also showed that the most potent antibody response was elicited against the C terminal region which contains a short lysine rich sequence aa362 381 KTFPPTEPKKDKKKKTDEAQ however the func tional role of this stretch remains unknown Van den Brink et al established two human anti N scFvs CR3018 and CR3009 which recognised a linear epitope RSAPRITFG aa11 19 and a nonlinear epitope respectively 18 In mice immunised with inactivated SARS CoV we identified one major epitope in the middle part aa110 210 which was recognised both by the SKOT 8 and SKOT 9mAb 10 whereas He et al identified one major epitope adjacent to the N terminal region aa76 101 in addition to epitopes in the C terminal and middle regions 44 Together these mapping studies of the SARS CoV N protein suggest that there are three major epitopes localised to the N terminal middle and C terminal regions respectively which are responsible for the potent immune response in both humans and mice Envelop E protein The small E protein is a 76aa residue long protein involved in the envelop mor phogenesis As far as we know there is only one mAb against this protein in humans 16 and this E protein antibody is detected in SARS conva lescent patient sera 23 122 Y Tsunetsugu Yokota et al Copyright 2006 John Wiley 16 117 131 Matrix M protein The M membrane glycopro tein is a 221aa residue long integral membrane protein which is functionally involved in the bud ding of virions from cells Pang et al reported that rabbit antiserum raised against recombinant M protein has a potent neutralising ability in vitro suggesting that the M protein could be one of the target proteins chosen for the vaccine develop ment 45 Wang et al reported that the synthetic peptide M137 aa137 158 is one of the most immunogenic regions of the SARS CoV structural proteins 24 Recently Zhong et al reported a sys tematic search for the viral epitopes by a bio panning of the M13 phage display dodecapeptide library using antibodies found in plasma samples of convalescent SARS patients 46 They determined the continuous viral epitopes including an epitope rich region on the S2 M and E proteins This contig uous epitope map of SARS CoV would greatly help to develop an effective vaccine for SARS CoV Antibody cross reactivity to OC43 and 229E We confirmed the absence of cross reactivity in the anti N mAbs SKOT 8 and SKOT 9 to human coronaviruses HCoV OC43 and HCoV 229E by ELISA 10 and IFA Figure 2 However at the polyclonal level there are reports of cross reactiv ity between SARS and other human coronaviruses Wo et al reported false positive results in their recombinant SARS CoV N protein based ELISA assay mainly due to the presence of cross reactive Figure 2 Immuno fluorescent assay showing the absence of cross reactivity with anti SARS CoV S protein mAb SKOT 3 and anti N protein mAb SKOT 8 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 columns and SKOT 8 middle column and polyclonal anti BCV CCV right columns Severe acute respiratory syndrome coronavirus 123 Copyright 2006 John Wiley 16 117 131 antibodies to SARS CoV N protein in the sera 47 They found that 3 out of 21 and 1 out of 7 sera from convalescent patients from HCoV OC43 and HCoV 229E respectively contained antibo dies cross reactive with the SARS CoV N protein suggesting the presence of a common epitope s in these human CoV N proteins The sera from convalescent patients however did not react with SARS C