【病毒外文文献】2005 Identification of Immunodominant Epitopes on the Membrane Protein of the Severe Acute Respiratory Syndrome-Associat

JOURNAL OF CLINICAL MICROBIOLOGY Aug 2005 p 3718 3726 Vol 43 No 8 0095 1137 05 08 00H110010 doi 10 1128 JCM 43 8 3718 3726 2005 Copyright 2005 American Society for Microbiology All Rights Reserved Identification of Immunodominant Epitopes on the Membrane Protein of the Severe Acute Respiratory Syndrome Associated Coronavirus Yuxian He 1 Yusen Zhou 2 Pamela Siddiqui 1 Jinkui Niu 1 and Shibo Jiang 1 Viral Immunology Laboratory Lindsley F Kimball Research Institute New York Blood Center New York New York 10021 1 and Department of Molecular Biology Beijing Institute of Microbiology and Epidemiology Beijing 100071 Peoples Republic of China 2 Received 29 December 2004 Returned for modification 20 February 2005 Accepted 25 April 2005 Similar to other coronaviruses the membrane M protein of severe acute respiratory syndrome associated coronavirus SARS CoV is a major transmembrane glycoprotein with multiple biological functions To date limited information is available about its antigenic properties In this study we identified two major immu nodominant epitopes on the M protein located in the extreme N terminal region residues 1 to 31 and the interior C terminal region residues 132 to 161 respectively by Pepscan analyses against convalescent phase sera from SARS patients and antisera from virus immunized mice and rabbits Synthetic peptides M1 31 derived from the N terminal epitope and M132 161 derived from the C terminal epitope were highly reactive with all of the convalescent phase sera from 40 SARS patients but not with 30 control serum samples from healthy blood donors suggesting their potential application for serologic diagnosis of SARS We showed that both peptides M1 31 and M132 161 were able to induce high titers of antibody responses in the immunized rabbits highlighting their antigenicity and immunogenicity These findings provide important information for developing SARS diagnostics and vaccines In November 2002 a new infectious pneumonia now known as severe acute respiratory syndrome SARS emerged in China and rapidly spread to 29 countries 19 25 34 More than 8 000 people were infected and ca 900 died during the outbreak in 2003 www who int A novel coronavirus SARS CoV was identified as the etiological agent of SARS and its genome was subsequently characterized 6 17 22 28 Al though the global outbreak of SARS was contained serious concerns remain over its reemergence in the future 9 De velopment of reliable diagnostics and vaccines is still a priority to control a new SARS epidemic Phylogenetic analyses demonstrate that SARS CoV is dis tinct from the three known antigenic groups of coronaviruses 22 28 With a genomic organization similar to those of other coronaviruses the large positive stranded RNA genome of SARS CoV encodes four major viral structural proteins the spike S membrane M envelope E nucleocapsid N proteins The M gene together with the genes for the other structural proteins is located in the 3H11032 one third of the viral genome downstream from the S gene and upstream from the N gene and encodes a glycoprotein with a predicted length of 221 amino acids Recently a number of studies suggest that the S protein of SARS CoV is a promising antigen for developing SARS vaccines since it can mediate protective immunity 1 3 12 42 and that the N protein may serve as an ideal antigen for SARS diagnosis since it can induce an appreciable antibody response in infected SARS patients 14 16 20 30 However little information is available on the immune response to the M protein of SARS CoV The M protein of coronavirus is the most abundant glycop rotein in the virus particle 29 The interaction between the M protein and the S protein as well as the N protein is essential for viral assembly and budding 29 The structure of M pro tein is characterized as having three domains a short N ter minal ectodomain a triple spanning transmembrane domain and a large interior C terminal domain It was previously dem onstrated that the M proteins of coronaviruses were able to induce antibody responses in hosts infected by coronavirus or immunized by attenuated recombinant virus expressing the M protein 8 18 26 35 39 Monoclonal antibodies to the M protein of mouse hepatitis virus a mouse coronavirus could neutralize virus infectivity in vitro 5 10 and protect animals against lethal virus challenge in vivo 10 These features make the M protein an attractive target for developing diagnostic tests and subunit vaccines 7 8 11 18 24 26 35 37 39 although its antigenic determinants remain to be defined Computer aided analyses suggest that the M protein of SARS CoV has a structure similar to the M proteins of other coro naviruses 22 28 It contains a short N terminus in the exterior of the virion residues 1 to 14 three transmembrane helices residues 15 to 37 50 to 72 and 77 to 99 and a 121 amino acid C terminal region inside the virus particle Fig 1 In the present study we sought to identify the immunodominant epitopes on the M protein of SARS CoV MATERIALS AND METHODS Synthetic peptides A set of 30 overlapping peptides spanning the entire sequence of the M protein was obtained through the AIDS Research and Ref erence Reagent Program Division of AIDS National Institute of Allergy and Infectious Disease National Institutes of Health These overlapping peptides range from 15 to 20 amino acids in length A set of peptides in Table 1 was synthesized by a standard solid phase Fmoc 9 fluorenylmethoxy carbonyl method in the MicroChemistry Laboratory of the New York Blood Center Peptides were purified to homogeneity purity of H1102295 by high performance liquid chromatography and identified by laser desorption mass spectrometry Corresponding author Mailing address Lindsley F Kimball Re search Institute New York Blood Center 310 East 67th St New York NY 10021 Phone 212 570 3058 Fax 212 570 3099 E mail SJiang NYBloodcenter org 3718 on March 12 2015 by PORTLAND STATE UNIV http jcm asm org Downloaded from Serum specimens from SARS patients Serum samples were collected from 40 convalescent phase SARS patients 30 to 60 days after onset of illness during the SARS outbreak in Beijing in 2003 The diagnostic criteria for SARS CoV infection followed the clinical description of SARS released by the World Health Organiza tion All of the sera were verified to be positive for SARS CoV by immunofluores cence assay and enzyme linked immunosorbent assay ELISA using commercially available diagnostic kits Beijing Genomics Institute Beijing Peoples Republic of China Sera from 30 healthy blood donors were used as controls Preparation of inactivated SARS CoV SARS coronavirus strain BJ01 acces sion number AY278488 was used as viral source and propagated in Vero E6 cells 27 The infected cells were harvested and completely lysed by three cycles of freeze thaw H9252 Propiolactone was then added to the lysates at 1 2 000 ratio followed by incubation at 37 C for 2 h The inactivated virus was centrifuged at 10 000 rpm for 20 min After removal of the cell debris the supernatants were desalted with Sephadex G 50 concentrated with PEG 8000 and filtrated with Sepharose CL 2B sequentially The inactivated SARS CoV in the final prepara FIG 1 Schematic diagram of SARS CoV M protein The M protein contains a short N terminus in the exterior of the virion residues 1 to 14 three transmembrane helices residues 15 to 37 50 to 72 and 77 to 99 and a 121 amino acid C terminal region inside the virus particle FIG 2 Mapping of immunodominant epitopes on the M protein of SARS CoV by ELISA A Antibodies specific for SARS CoV in the convalescent phase sera from 40 SARS patients were measured by commercial diagnostic kit in which the mixture of proteins purified from viral lysates of SARS CoV was used as coating antigen B Pepscan analysis against the convalescent phase sera from 40 SARS patients with a set of 30 overlapping peptides that span the entire sequence of the M protein as coating antigens Sera tested at 1 50 dilution were considered positive when the A 450 values were above the cutoff value mean A 450 value of sera from health blood donors plus three standard deviations VOL 43 2005 IMMUNODOMINANT EPITOPES ON THE SARS CoV M PROTEIN 3719 on March 12 2015 by PORTLAND STATE UNIV http jcm asm org Downloaded from tion with H1102295 purity analyzed by high pressure liquid chromatography was confirmed by observing the coronavirus like particles under an electron micro scope and by determining the reactivity with convalescent phase sera of SARS patients in Western blots 33 Immunizations BALB c mice and New Zealand White rabbits were immu nized intradermally with 10 and 30 H9262g respectively of purified H9252 propiolactone inactivated viral particles as immunogen in the presence of Freund complete adjuvant and boosted with freshly prepared emulsion of the immunogen and Freund incomplete adjuvant at 2 wk intervals Preimmune sera preimmune were collected before the primary immunization and antisera were collected 5 days after the third boost immunization Rabbit antisera directed against pep tides M1 31 and M132 161 were produced at Covance Research Products Inc Denver PA by using their standard protocols Briefly New Zealand White rabbits were immunized intradermally with 250 H9262g of purified peptides resus pended in phosphate buffered solution pH 7 2 in the presence of Freund complete adjuvant and boosted with freshly prepared emulsion of the immuno gen and Freund incomplete adjuvant at 3 week intervals Rabbit antisera were collected 10 days after each boost Sera were kept at 4 C before use ELISA The reactivities of the M protein derived peptides with the convales cent phase sera from SARS patients and the serum samples from mice and rabbits immunized with the inactivated viral particles or synthetic peptides were determined respectively by ELISA Briefly each peptide 10 H9262g ml was coated onto wells of a 96 well microtiter plate Corning Costar Acton MA in 0 1 M carbonate buffer pH 9 6 at 4 C overnight After blocking with 2 nonfat milk the plate was incubated with corresponding antisera at indicated dilution at 37 C for 1 h and then washed three times with PBS containing 0 1 Tween 20 Bound antibodies were detected with horseradish peroxidase conjugated goat anti hu man immunoglobulin G IgG or anti mouse IgG or anti rabbit IgG Zymed South San Francisco CA accordingly at 37 C for 1 h followed by washing The reaction was visualized by addition of the substrate 3 3H11032 5 5H11032 tetramethylbenzi dine and the absorbance at 450 nm A 450 was measured by an ELISA plate reader Tecan US Research Triangle Park NC RESULTS Mapping of immunodominant epitopes on the M protein in SARS patients A total of 40 serum samples from convalescent FIG 3 Mapping of immunodominant epitopes on the M protein of SARS CoV by Pepscan analysis against antisera from mice A and rabbits B immunized with inactivated SARS CoV A set of 30 overlapping peptides that span the entire sequence of the M protein were used as coating antigens in ELISA Antisera were tested at 1 100 dilutions TABLE 1 Peptides overlapping the immunodominant epitopes of M protein Peptide Sequence N terminal peptides M1 16 MADNGTITVEELKQLL M7 23 ITVEELKQLLEQWNLVI M1 20 MADNGTITVEELKQLLEQWN M1 23 MADNGTITVEELKQLLEQWNLVI M4 26 NGTITVEELKQLLEQWNLVIGFL M1 31 MADNGTITVEELKQLLEQWNLVIGFLFLAWI C terminal peptides M140 157 GAVIIRGHLRMAGHPLGR M132 161 LMESELVIGAVIIRGHLRMAGHPLGRCDIK M164 180 PKEITVATSRTLSYYKL M158 185 CDIKDLPKEITVATSRTLSYYKLGASQR 3720 HE ET AL J CLIN MICROBIOL on March 12 2015 by PORTLAND STATE UNIV http jcm asm org Downloaded from SARS patients were collected to characterize the immunoge nicity of SARS CoV M protein All samples were verified to be positive to SARS CoV as detected by ELISA with commer cially available diagnostic kits with the mixture of proteins purified from viral lysates of SARS CoV as the coating antigen Fig 2A To map the immunodominant epitopes on the M protein a set of 30 overlapping peptides that span the entire sequence of the M protein were used as coating antigens in ELISA and serum samples were tested against each of the peptides As shown in Fig 2B whereas most of the peptides did not significantly react with any of the serum samples from SARS patients the peptide 7 23 17 mer from the N terminus and the peptide 140 157 18 mer from the C terminal interior region reacted respectively with ca 70 of serum samples This result indicates that these two sites on the M protein contain the major immunodominant epitopes that induce an tibody responses in humans In addition the peptide 164 180 was reactive with ca 40 of serum samples suggesting this site is a minor immunodominant epitope in the SARS patients Mapping of immunodominant epitopes on the M protein in immunized animals Inactivated SARS CoV a major candi date SARS vaccine can induce robust antibody responses in immunized animals To map the antigenic sites of M protein two mice and two rabbits were immunized with the inactivated SARS CoV as immunogen and their antisera were tested against each of the overlapping peptides derived from the entire M protein Interestingly both mouse and rabbit antisera significantly reacted with the N terminal peptide 7 23 and C terminal peptide 140 157 that were identified as immunodom inant sites by human SARS sera Fig 3 This result suggests that these two sites also function as immunodominant epitopes in immunized mice and rabbits highlighting the immunogenic ity of M protein In addition peptide 97 111 reacted weakly with the mouse and rabbit antisera but not with sera of SARS patient suggesting that this region may serve as a specific antigenic site in animals Identification of highly immunoreactive peptides for sero logic diagnosis of SARS Pepscan analyses against the conva lescent phase sera from SARS patients and the antisera from immunized animals revealed the N terminal and C terminal immunodominant epitopes on the M protein To further char acterize their epitopic structure we designed a set of ten pep tides that overlap with the N terminal or C terminal antigenic sequence as probes Table 1 These peptides were used as coating antigens in ELISA to test their reactivity with 18 ran domly selected serum samples from SARS patients Strikingly two longer peptides M1 31 overlapping with the N terminal epitope and M132 161 overlapping with the C terminal epitope were highly reactive with all of the SARS serum sam ples tested Table 2 The peptides M1 23 and M4 26 had better reactivities with SARS sera than the peptide M7 23 which was used in Pepscan analyses suggesting that the ex tended sequences at both N and C termini of the M7 23 Table 1 are important to the constitution of the N terminal immu nodominant epitope however the reactivity of peptides was significantly improved by adding more residues at the C ter minus of the peptide These data suggest that both of the N terminal and C terminal immunodominant epitopes in the M protein may require longer sequences to maintain the in tegrity of their antigenic conformations The antigenicity of synthetic peptides was further evaluated by measuring their reactive titers to antiserum samples As shown in Fig 4 the peptides M1 31 and M132 161 reacted TABLE 2 Reactivity of convalescent sera from SARS patients with N terminal or C terminal peptides a Serum Viral proteins Reactivity A 450 with N terminal peptides C terminal peptides M1 16 M7 23 M1 20 M1 23 M4 26 M1 31 M140 157 M132 161 M158 185 SARS sera No 1 2 54 0 09 0 57 0 68 1 19 2 19 2 56 0 60 0 69 0 31 No 2 0 85 0 03 0 94 1 55 2 51 2 12 1 99 0 01 0 67 0 07 No 3 1 90 0 44 0 56 0 69 1 37 1 11 2 58 0 95 1 13 0 41 No 4 1 62 0 03 0 21 0 06 0 46 0 62 2 36 0 54 0 76 0 15 No 5 1 68 0 58 0 32 0 42 0 69 0 47 2 48 1 10 1 66 0 21 No 6 1 57 0 12 0 88 0 58 1 10 0 77 2 44 0 01 0 90 0 02 No 7 2 22 0 08 0 04 0 14 0 32 0 35 2 71 0 48 0 76 0 28 No 8 1 50 0 07 0 25 0 10 0 27 0 34 1 44 0 47 0 73 0 20 No 9 1 07 0 11 0 10 0 18 0 34 0 06 2 12 0 07 1 81 0 10 No 10 2 21 0 26 0 30 0 28 0 27 0 25 0 85 0 68 1 16 0 29 No 11 2 98 0 13 0 86 0 09 0 32 0 23 1 61 0 06 0 87 0 17 No 12 2 96 0 07 0 04 H110020 01 0 03 0 11 1 36 0 34 0 67 0 09 No 13 1 68 0 07 0 11 0 10 0 14 0 06 1 41 0 06 0 85 0 07 No 14 1 45 0 08 0 32 0 04 0 03 0 01 2 78 0 07 0 95 0 07 No 15 1 42 0 02 0 00 0 16 0 57 0 34 2 48 0 52 0 60 0 14 No 16 0 98 0 08 0 03 0 08 0 06 0 05 1 14 H110020 04 0 78 0 45 No 17 1 64 0 44 0 45 0 77 0 80 1 31 1 17 1 20 0 78 0 27 No 18 1 48 0 19 0 12 0 07 0 09 0 15 2 54 0 50 0 59 0 15 Control sera C 1 0 03 0 07 0 07 0 80 0 06 0 13 0 09 0 09 0 03 0 09 C 2 0 08 0 08 0 09 0 11 0 07 0 09 0 05 0 06 0 12 0 08 a Peptides were used at 10 H9262g ml and sera were tested at a 1 50 dilution Positive values are in boldface VOL 43 2005 IMMUNODOMINANT EPITOPES ON THE SARS CoV M PROTEIN 3721 on March 12 2015 by PORTLAND STATE UNIV http jcm asm org Downloaded from with seven serum samples from SARS patients with mean endpoint titers of 1 3 628 and 1 1 020 respectively The pep tide M1 31 reacted with the mouse or rabbit antisera with mean titers at 1 1 460 or 1 1 788 respectively whereas the peptide M132 161 reacted with the mouse or rabbit antisera with mean titers at 1 6 400 or 1 8 505 respectively Obviously peptide M1 31 had higher reactive titers with SARS patient sera but lower titers with mouse or rabbit antisera than M132 161 had In contrast the M132 161 strongly reacted with the animal antisera but not the patient sera This result suggests that the N terminal epitope is more immunogenic in infected patients but less immunogenic in immunized animals than the C terminal epitope To develop immunoassays for SARS diagnosis peptides M1 31 and M132 161 were used respectively as antigens for ELISA to detect site specific antibodies in serum samples from SARS patients As shown in Fig 5 both peptides significantly reacted with each of the 40 patient sera but none of the control sera from healthy blood donors Notably the peptide M1 31 had higher reactivity in ELISA than peptide M132 161 FIG 4 Immunoreactivity of synthetic peptides M1 31 and M132 161 derived from the N terminal or C terminal immunodominant epitopes on the M protein with the convalescent phase sera from 40 SARS patients and antisera from immunized animals Serum samples were tested as a series of twofold dilutions by ELISA 3722 HE ET AL J CLIN MICROBIOL on March 12 2015 by PORTLAND STATE UNIV http jcm asm org Downloaded from This result further indicates that these two peptides have po tential to be developed as specific diagnostic antigens Synthetic peptides induced potent antibody responses in the rabbits To evaluate the immunogenicity of synthetic peptides derived from the immunodominant epitopes of M protein M1 31 and M132 161 were used as immunogens for the im munization of rabbits Strikingly the rabbits developed high titers of antibody responses after the first boosted immuniza tion for both peptides and retained similar levels after the second and the third boosts After the first boost the endpoint titers of antisera from two rabbits immunized with M1 31 were 1 102 400 and 1 1 638 400 respectively Fig 6A and B whereas those against M131 161 were 1 26 214 400 and 1 6 553 600 respectively Fig 6C and D This result indicated that both the N terminal and C terminal peptides were highly immunogenic in the immunized rabbits however the peptide M132 161 induced more potent antibody responses than the peptide M1 31 a finding consistent with the site specific anti body responses in the rabbits immunized with the inactivated SARS CoV DISCUSSION Postgenomic characterization of SARS CoV is important for exploring the mysteries of SARS We sought to character ize the antigenicity and immunogenicity of SARS CoV since this work is critical for developing effective SARS diagnostics and vaccines Recently we demonstrated that the S and N proteins two major structural proteins of SARS CoV contain several immunodominant sites that induce appreciable anti body responses during viral infec