【病毒外文文献】2005 Identification of a critical neutralization determinant of severe acute respiratory syndrome (SARS)-associated coro

SARS a b a b b 2C5 but not 1A5 was able to block binding of the RBD to angiotensin converting enzyme 2 ACE2 the functional receptor on targeted cells These data provide important information for understanding the antigenicity and immunogenicity of SARS CoV and for designing Similar to other coronaviruses SARS CoV features a large positive stranded RNA genome that harbors open reading The high rate of recovery from acute illness of SARS in the absence of effective medical therapy and the low rate of re infection by SARS CoV suggest that protective immunity is that sera from Virology 334 2005 SARS vaccines D 2005 Elsevier Inc All rights reserved Keywords SARS CoV Spike protein Receptor binding domain Neutralizing antibodies Monoclonal antibodies Vaccines Introduction The global emergency of severe acute respiratory syndrome SARS was caused by a new coronavirus SARS CoV Drosten et al 2003 Ksiazek et al 2003 Marra et al 2003 Peiris et al 2003 Rota et al 2003 frames ORFs encoding a large polyprotein required for virus replication four structural proteins spike S envelop E membrane M and nucleocapsid N and eight additional polypeptides of unknown function Marra et al 2003 Rota et al 2003 SARS CoV infection can trigger humoral and cellular immune responses against viral proteins in humans Yuxian He Qingyu Zhu T Shuwen Liu Yusen Zhou Baoan Yang Jiaming Li b Shibo Jiang a T a Viral Immunology Laboratory Lindsley F Kimball Research Institute New York Blood Center 310 East 67th Street New York NY 10021 USA b Beijing Institute of Microbiology and Epidemiology Beijing 100071 China Received 3 December 2004 returned to author for revision 4 January 2005 accepted 26 January 2005 Abstract The spike S protein of severe acute respiratory syndrome associated coronavirus SARS CoV is not only responsible for receptor binding but also a major antigenic determinant capable of inducing protective immunity In this study we demonstrated that the receptor binding domain RBD of S protein is an important immunogenic site in patients with SARS and rabbits immunized with inactivated SARS CoV Serum samples from convalescent SARS patients and immunized rabbits had potent neutralizing activities against infection by pseudovirus expressing SARS CoV S protein Depletion of RBD specific antibodies from patient or rabbit immune sera by immunoadsorption significantly reduced serum mediated neutralizing activity while affinity purified anti RBD antibodies had relatively higher potency neutralizing infectivity of SARS pseudovirus indicating that the RBD of S protein is a critical neutralization determinant of SARS CoV during viral infection and immunization Two monoclonal antibodies 1A5 and 2C5 targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS CoV Both 1A5 and 2C5 possessed potent neutralizing activities although they directed against distinct conformation dependant epitopes as shown by ELISA and binding competition assay We further demonstrated that Identification of a critical neutralization respiratory syndrome SARS associated for designing 0042 6822 see front matter D 2005 Elsevier Inc All rights reserved doi 10 1016 j virol 2005 01 034 T Corresponding authors Fax 1 212 570 3099 E mail address SJiang NYBloodcenter org S Jiang determinant of severe acute coronavirus Importance vaccines 74 82 achievable Recent studies have demonstrated convalescent phase SARS patients contain high titers of neutralizing antibodies against SARS CoV Nie et al 2004 experiments revealed that protection was mediated by neutralizing antibodies Subbarao et al 2004 Yang et al RBD in humans All the sera were positive for SARS CoV as detected by ELISA with commercially available diag nostic kits in which the mixture of proteins purified from viral lysates of SARS CoV was used as coating antigen data not shown To determine antibodies specific for the RBD of S protein in the SARS convalescent sera we used recombinant RBD C9 as a coating antigen in ELISA As shown in Fig 1 all of the 40 convalescent sera from SARS patients reacted significantly with RBD C9 whereas none of the sera from healthy blood donors was reactive to this antigen suggesting that the RBD of SARS CoV S protein is highly immunogenic during virus infection Isolation of RBD specific antibodies from convalescent sera of SARS patients The RBD Fc fusion protein was used to isolate RBD specific antibodies by immunoaffinity chromatography from the convalescent sera of SARS patients Serum samples from four SARS patients SARS A D pre 334 2005 74 82 75 2004 and protection can be achieved by sole adminis tration of neutralizing monoclonal antibodies specific for S protein Traggiai et al 2004 Furthermore vaccination of animals with recombinant viruses such as attenuated vaccinia virus MVA and parainfluenza virus BHPIV3 that express S protein can elicit neutralizing antibodies to protect animals against SARS CoV challenge Bisht et al 2004 Buchholz et al 2004 Bukreyev et al 2004 Infection by pseudovirus expressing the SARS CoV S protein can be effectively neutralized by convalescent sera from SARS patients Nie et al 2004 Simmons et al 2004 These data suggest that the S protein of SARS CoV is a promising candidate for developing SARS vaccines The S protein of SARS CoV a large class I trans membrane glycoprotein consisting of S1 domain residues 15 680 and S2 domain residues 681 1255 mediates attachment of virions to susceptible cells and fusion of the viral and cellular membranes Hofmann and Pohlmann 2004 Holmes 2003 A 193 amino acid small fragment residues 318 510 in the S1 region was characterized as the minimal receptor binding domain RBD of SARS CoV Babcock et al 2004 Wong et al 2004 Xiao et al 2003 RBD mediates S protein binding to angiotensin converting enzyme 2 ACE2 the functional receptor for SARS CoV on susceptible cells Dimitrov 2003 Li et al 2003 Prabakaran et al 2004 Wang et al 2004 Most recently we demonstrated that the recombinant RBD of SARS CoV S protein could induce potent neutralizing antibodies He et al 2004a 2004b In this report we showed that the RBD of S protein is a critical neutralization determinant of SARS CoV in SARS patients and SARS CoV immunized rabbits We further identified two neutralization epitopes in the RBD by monoclonal antibodies MAbs isolated from mice immunized with inactivated SARS CoV Results The RBD of S protein is an immunogenic site of SARS CoV in infected patients It has been demonstrated that the RBD of S protein is highly immunogenic in mice and rabbits immunized with inactivated SARS CoV He et al 2004b In this study 40 Simmons et al 2004 The S protein of SARS CoV like those of other coronaviruses has been shown to be a major antigenic determinant that induces neutralizing antibodies and protective immunity Bisht et al 2004 Buchholz et al 2004 Bukreyev et al 2004 Yang et al 2004 A DNA vaccine encoding the S protein induced SARS CoV neutralization and protective immunity in mice Yang et al 2004 Depletion of T cells and antibody transfer Y He et al Virology serum samples were collected from SARS patients at convalescent phase to investigate the immunogenicity of screened for high levels of reactivity against RBD C9 and a control serum from health blood donor were ad sorbed on resins immobilized with RBD Fc fusion protein and bound antibodies anti RBD were eluted with pH 2 5 buffer The efficiencies of depletion and recovery of the RBD specific antibodies were monitored by measuring the reactivity of the starting human sera the corresponding flowthroughs and eluted antibody fractions by ELISA against the RBD C9 and S1 C9 As shown in Fig 2A the reactivity of anti RBD in the sera of SARS patients was efficiently removed by RBD Fc affinity column The eluted anti RBD antibodies could significantly react with RBD C9 All samples corresponding to the starting sera flowthroughs anti RBD depleted and eluates anti RBD Fig 1 Measurement of the RBD specific antibodies in sera from convalescent phase SARS patients by ELISA Recombinant RBD C9 was used for coating plates and the sera from 40 SARS patients and 30 healthy blood donors were tested at 1 50 dilution Sera were considered positive when the optical density values were above the cutoff value the mean absorbance at 450 nm of sera from healthy blood donors plus three standard deviations 334Y He et al Virology76 antibodies were reactive to the S1 C9 in ELISA Fig 2B These suggest that the convalescent sera of SARS patients contain abundant antibodies specific for S protein and the RBD specific antibodies can be specifically isolated by immunoaffinity chromatography The RBD of S protein is a potent inducer of neutralizing antibodies in infected patients Entry of SARS CoV into target cells is initiated by binding of its S protein via RBD in the S1 region to ACE2 a cellular receptor It has been shown that a fusion protein RBD Fc containing the RBD of S protein is a potent inducer of neutralizing antibodies against SARS CoV in immunized animals He et al 2004a It is interesting to know whether the RBD of S protein can mediate antibody responses responsible for virus neutralization in infected SARS patients Therefore the neutralizing activity of the samples of the starting sera flowthroughs and eluates was determined using SARS pseudovirus Strikingly the neu tralizing activity of patient sera was significantly reduced after depletion of anti RBD antibodies The anti RBD antibodies in the eluates possessed higher potency than Fig 2 Isolation of RBD specific antibodies from sera of convalescent phase SARS patients by immunoaffinity chromatography Serum samples were adsorbed to RBD Fc columns and bound antibodies were eluted at pH 2 5 The patient sera flowthroughs and eluates were tested respectively against RBD C9 A and S1 C9 B at 1 50 dilution by ELISA One serum sample from a healthy blood donor was used as control the antibodies in the flowthroughs to neutralize SARS pseudovirus Fig 3 and Table 1 suggesting that more than 50 neutralizing activity in the convalescent sera of SARS patients was contributed by the RBD specific antibodies In contrast the serum samples including the starting sera flowthroughs and eluates from the healthy blood donor had no neutralizing activities to same pseudovirus data not shown These data indicate that the RBD of S protein is an important inducer of neutralizing antibodies during viral infection The RBD of S protein is a critical antigenic site to induce neutralizing antibodies in rabbits immunized with inactivated SARS CoV We previously demonstrated that the inactivated SARS CoV was able to induce neutralizing antibodies in the immunized rabbits He et al 2004b To further elucidate neutralization determinants of the SARS CoV the RBD specific antibodies were isolated by immunoaffinity chro matography from four rabbit antisera A D Similarly the reactivity of anti RBD antibodies in the rabbit antisera could be efficiently depleted by RBD Fc affinity column since the flowthroughs did not bind to RBD Fc while the eluted anti RBD antibodies significantly reacted with the RBD Fc Fig 4A Samples corresponding to the starting sera flow throughs anti RBD depleted sera and eluates containing anti RBD antibodies had similar reactivity with the S1 C9 Fig 4B The neutralizing activity of anti RBD antibody depleted rabbit antisera was much lower than the starting sera while anti RBD antibodies possessed more potent activity to neutralize SARS pseudovirus Fig 4Cand Table 1 These data suggest that the RBD of S protein is an effective inducer of neutralizing antibodies in virus immunized rabbits Isolation and characterization of MAbs against the RBD of S protein It has been shown that inactivated SARS CoV can induce high titers of antibodies specific for the RBD of S protein in immunized mice He et al 2004b To identify neutralization epitopes of SARS CoV two MAbs 1A5 and 2C5 targeting at the RBD of S protein were isolated from mice immunized with inactivated SARS CoV Epitope specificities of 1A5 IgG1 n and 2C5 IgG1 n were initially determined by ELISAs using RBD Fc DTT reduced RBD Fc S1 C9 and DTT reduced S1 C9 as coating antigens Figs 5A and B Both MAbs were reactive with native RBD Fc and S1 C9 but not DTT reduced RBD Fc and S1 C9 indicating that they were directed against disulfide bond dependent conformational epitopes expressed on the RBD of S protein These two MAbs did not compete each other in an ELISA based 2005 74 82 binding competition assay Fig 5C suggesting their epitopes locate at different sites in the RBD sera of sera flowthroughs and eluates respectively at a series of 2 fold dilutions was Y He et al Virology 334 Fig 3 Neutralizing activity of the RBD specific antibodies isolated from the D Inhibition of SARS pseudovirus infectivity in 293T ACE2 cells by patient determined and the percentage of neutralization was calculated for each sample RBD Fc could efficiently bind to ACE2 expressed on 293T ACE2 cells as measured by flow cytometry We tested whether the RBD specific MAbs inhibit binding of RBD Fc to cell associated ACE2 As shown in Fig 6 2C5 but not 1A5 was able to block RBD Fc binding to 293T ACE2 cells This result suggests that 2C5 recognizes an epitope that may overlap with the receptor binding sites in the S protein while 1A5 directs against a distinct conformation dependant epitope that is not involve in receptor binding With a single cycle infection assay we tested the neutraliz ing activities of these two MAbs against SARS pseudovirus Strikingly 1A5 and 2C5 potently neutralized SARS pseudovirus with 50 neutralization dose ND 50 at 0 048 Ag ml and 0 097 Ag ml respectively Fig 7 suggesting Table 1 Neutralizing activity of serum samples against SARS pseudovirus Sera sample Neutralization titer ND 50 a Starting sera Flowthroughs Eluates Human sera SARS A 2812 945 1694 SARS B 4999 1718 2670 SARS C 3031 796 1822 SARS D 2614 907 1052 Rabbit sera A 2202 750 1250 B 1966 845 1125 C 2142 772 1050 D 1868 698 980 a ND 50 Titer of a serum sample to yield 50 neutralization SARS patients A SARS A B SARS B C SARS C and D SARS 2005 74 82 77 that both conformation dependant epitopes in the RBD of S protein can elicit potent neutralizing antibodies Discussion The S protein of SARS CoV is able to induce protective immunity in immunized animals Bishtetal 2004 Buchholz et al 2004 Bukreyev et al 2004 Yang et al 2004 Using a set of overlapping peptides that cover the entire sequence of S protein we have identified several immunodominant domains within S protein However none of these domains modeled by linear peptides were able to elicit neutralizing antibodies He et al 2004c In contrast the RBD residues 318 510 in the S1 region of S protein can induce highly potent neutralizing antibodies against the SARS CoV He et al 2004a 2004b Here we have further demonstrated that the RBD of SARS CoV S protein is an immunogenic site to induce antibody responses in SARS CoV infected patients and immunized rabbits With an immunoabsorption assay we have demonstrated that anti RBD antibodies function as a main population of neutraliz ing antibodies suggesting that the RBD of S protein is an important neutralization determinant of SARS CoV The first essential step of coronavirus infection is the interaction of S protein via the RBD with specific cellular receptor The RBDs on the S proteins of other coronavi ruses such as mouse hepatitis virus MHV transmissible gastroenteritis virus TGEV and human coronavirus SARS CoV Therefore the RBD of S protein may serve as an important target site for developing SARS vaccines and immunotherapeutics Inactivated SARS CoV has been used as one of the major vaccine candidates and is currently being tested in clinical trial in China Marshall and Enserink 2004 However caution should be taken in using the inactivated SARS CoV 334 2005 74 82Y He et al Virology78 HCoV 229 also contain major antigenic determinants capable of inducing neutralizing antibodies Bonavia et al 2003 Godet et al 1994 Kubo et al 1994 We previously demonstrated that inactivated SARS CoV vac cine was capable of inducing high titers of RBD specific antibodies that block receptor binding and virus entry He et al 2004b In the present study two neutralization epitopes in the RBD of S protein have been identified with MAbs isolated from mice immunized with inactivated asvaccinesinceitcontainsanumberofviralcomponentsthat may induce harmful immune and or inflammatory responses Enserink 2004 He et al 2004c Oba 2003 Wang and Lu Fig 4 Isolation and characterization of RBD specific antibodies from rabbit antisera by immunoaffinity chromatography Reactivities of rabbit antibodies with RBD Fc A and S1 C9 B were tested at 1 50 dilution by ELISA C Neutralizing activity of the RBD specific antibodies isolated from rabbit antisera Inhibition of SARS pseudovirus infection in 293T ACE2 cells by rabbit antisera flowthroughs and eluates respectively at a series of 2 fold dilutions was determined and the percent neutralization was calculated for each sample Fig 5 Characterization of the RBD specific MAbs The reactivities of MAbs against RBD Fc A and S1 C9 B were tested by ELISA MAbs were tested at 10 Ag ml and control sera were tested at 1 100 dilution C Inhibition of biotinylated MAbs binding to RBD Fc by RBD specific MAbs measured by competitive ELISA Competing MAbs were tested at 100 Ag ml 2004b Plasmids encoding a 193 amino acid fragment of RBD residues 318 510 linked to the Fc domain of human IgG1 designated RBD Fc or tagged with C9 designated RBD C9 and plasmid encoding residues 12 672 corresponding to the S1 domain of S protein tagged with C9 at the C terminus designated S1 C9 were kindly provided by Dr M Farzan at the Harvard Medical School Li et al 2003 Wong et al 2004 RBD Fc RBD C9 and S1 C9 proteins were expressed in 293T cells trans fected with the plasmids using Fugene 6 reagents Boehringer Mannheim Indianapolis IN according to the manufacturer s protocol Supernatants were harvested 72 h post transfection RBD C9 and S1 C9 were purified by affinity chromatography with anti C9 MAb 1D4 National Cell Culture Center Minneapolis MN RBD Fc was purified by Protein A Sepharose 4 Fast Flow 334 2005 74 82 79 2004 It was recently reported that an inactivated SARS CoV vaccine triggered an autoimmune response against the carbohydrate moieties in an abundant human serum glyco protein asialo orosomucoid Wang et al 2004 Recombi nant viruses or DNA vaccines that express full length S protein have also been considered as SARS vaccine candidates Bisht et al 2004 Buchholz et al 2004 Bukreyev et al 2004 Yang et al 2004 However the full length S protein also contains several linear immunodo minant domains that induce a high titer of non neutralizing antibodies Heetal 2004c Itisunclearwhetherornotthese non neutralizing antibodies may enhance SARS CoV infec tion or mediate harmful immune responses It was reported that vaccination of ferrets with vaccinia virus based SARS vaccine expressing full length S protein aggravated liver damage caused by SARS CoV infection Enserink 2004 Weingartl et al 2004 It has been demonstrated that the S protein of feline coronavirus FIPV expressed by recombi Fig 6 Inhibition of RBD Fc binding to ACE2 by MAbs Inhibitory activity of MAbs on RBD Fc binding to cell associated ACE2 expressed on 293T ACE2 cells was measured by flow cytometry RBD Fc and MAbs were used at 1 and 50 Ag ml respectively Y He et al Virology nant vaccinia can cause antibody dependant enhancement of disease if the vaccinated animals are subsequently infected with wild type virus Corapi et al 1992 Olsen et al 1993 Vennema et al 1990 Therefore we propose to use the RBD of SARS CoV S protein for developing an effective and safe subunit vaccine since the RBD of S protein is not only a functional domain that mediates virus receptor binding but also a critical neutralization determinant of SARS CoV If an effective RBD based SARS vaccine can be developed it may pro