【病毒外文文献】2005 Single Amino Acid Substitutions in the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Determine V

JOURNAL OF VIROLOGY Sept 2005 p 11638 11646 Vol 79 No 18 0022 538X 05 08 00H110010 doi 10 1128 JVI 79 18 11638 11646 2005 Copyright 2005 American Society for Microbiology All Rights Reserved Single Amino Acid Substitutions in the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Determine Viral Entry and Immunogenicity of a Major Neutralizing Domain Christopher E Yi Lei Ba Linqi Zhang David D Ho and Zhiwei Chen Aaron Diamond AIDS Research Center The Rockefeller University New York New York 10016 Received 16 March 2005 Accepted 29 June 2005 Neutralizing antibodies NAbs against severe acute respiratory syndrome SARS coronavirus SARS CoV spike S glycoprotein confer protection to animals experimentally infected with the pathogenic virus We and others previously demonstrated that a major mechanism for neutralizing SARS CoV was through blocking the interaction between the S glycoprotein and the cellular receptor angiotensin converting enzyme 2 ACE2 In this study we used in vivo electroporation DNA immunization and a pseudovirus based assay to functionally evaluate immunogenicity and viral entry We characterized the neutralization and viral entry determinants within the ACE2 binding domain of the S glycoprotein The deletion of a positively charged region SH9004 422 463 abolished the capacity of the S glycoprotein to induce NAbs in mice vaccinated by in vivo DNA electroporation Moreover the SH9004 422 463 pseudovirus was unable to infect HEK293T ACE2 cells To determine the specific residues that contribute to related phenotypes we replaced eight basic amino acids with alanine We found that a single amino acid substitution R441A in the full length S DNA vaccine failed to induce NAbs and abolished viral entry when pseudoviruses were generated However another substitution R453A abolished viral entry while retaining the capacity for inducing NAbs The difference between R441A and R453A suggests that the determinants for immunogenicity and viral entry may not be identical Our findings provide direct evidence that these basic residues are essential for immunogenicity of the major neutralizing domain and for viral entry Our data have implications for the rational design of vaccine and antiviral agents as well as for understanding viral tropism Severe acute respiratory syndrome SARS first identified in China in November 2002 is an emerging infectious disease caused by a novel coronavirus CoV variant SARS CoV 11 22 23 26 SARS CoV is highly transmissible in humans with a mortality rate near 10 10 25 28 Because of the threat of a reemerging epidemic much effort has been placed on the development of a prophylactic vaccine against the pathogenic SARS CoV 3 5 6 20 33 36 Initially it was first shown in a mouse model that passively transferred antibodies can prevent SARS CoV replication in the lung following intranasal viral challenge 29 Subsequently several groups reported that DNA and live virus adenovirus type 5 and modified vaccinia Ankara MVA vaccines expressing SARS CoV spike S gly coprotein were able to induce T cell and antibody responses 3 6 12 36 39 41 43 Moreover protective immunity was achieved using a DNA or MVA vaccine in a mouse model 3 36 Further observations show a correlate of protection me diated by neutralizing antibodies NAbs 3 36 These find ings are encouraging and indicate the potential for vaccine induced protective immunity in humans Shortly after SARS CoV was identified Li et al revealed that angiotensin converting enzyme 2 ACE2 was the func tional receptor for SARS CoV 24 The interaction between ACE2 and SARS CoV S glycoprotein was further elucidated to reveal the structure and function of ACE2 and of the viral envelope protein 27 ACE2 was found to interact with an independently folded receptor binding domain RBD a 193 residue fragment S 318 510 of the SARS CoV S protein 34 This 193 residue fragment alone exhibited potent antivi ral activity through blocking S protein mediated infection This finding suggests that the functional elements required to compete with SARS CoV binding to ACE2 are located within amino acids 318 to 510 In a separate study a single chain variable fragment 80R human monoclonal antibody MAb efficiently neutralized SARS CoV and inhibited syncytia for mation between cells expressing the S protein and those ex pressing ACE2 30 Since this antibody also blocked the in teraction between ACE2 and S glycoprotein the ACE2 binding site of the S glycoprotein has become an attractive target for vaccine design 30 Of the structural proteins that compose SARS CoV the S glycoprotein is probably the only significant target for neutral ization 4 We and others previously demonstrated that the receptor binding domain of the S protein contains a major neutralization determinant 6 15 16 which can induce potent NAbs that block SARS CoV replication in monkeys 6 Re gardless of the forms of vaccines tested e g DNA MVA and inactivated virus 3 16 35 36 a major neutralization deter minant is well exposed in vivo for inducing substantial levels of NAbs However specific mapping of the major neutralizing epitope is yet to be determined Here we delete a mainly positively charged region SH9004 422 463 in the receptor binding domain of the S glycoprotein and observe a loss in the ability Corresponding author Mailing address Aaron Diamond AIDS Research Center The Rockefeller University 455 1st Avenue 7th Floor New York NY 10016 Phone 212 448 5031 Fax 212 725 1126 E mail zchen adarc org These authors contributed equally to this work 11638 on March 7 2015 by GEORGIAN COURT UNIV http jvi asm org Downloaded from to induce potent NAbs as well as mediate viral entry More over these phenotypes are determined by positively charged residues in the deletion region Our findings have implications for SARS vaccine and antiviral design in addition to under standing the biological function of the viral envelope glycop rotein MATERIALS AND METHODS SARS CoV S glycoprotein mutagenesis The codon optimized full length SARS CoV S gene was cloned into pcDNA3 1 as a positive control pcDNA3 1 OPT9 S as previously described 6 Based on pcDNA3 1 OPT9 S we created a construct pCMV SH9004 422 463 by removing a 42 amino acid aa region H9004 422 463 within the ACE2 receptor binding region of the S gene Addition ally primers were constructed to mutate eight basic residues R426 K439 R441 R444 H445 K447 R449 and R453 to alanine A total of eight constructs mutants 1 through 8 was derived from the codon optimized full length S gene of SARS CoV in pcDNA3 1 Invitrogen Carlsbad CA using the QuickChange II XL Site Directed Mutagenesis kit Stratagene La Jolla CA Each of the mutated residues lies within the deletion region of pCMV SH9004 422 463 Each plasmid was purified with the GenElute Endotoxin free Plasmid Maxiprep kit Sigma Aldrich St Louis MO and resuspended in saline All plasmids were confirmed by sequence analysis through the DNA Sequencing Core Facility at the Aaron Diamond AIDS Research Center Expression of mutant S proteins 293T cells 5 H11003 10 5 cells per well in 6 well plates were transfected Superfect Transfection Reagent QIAGEN Valencia CA with 2 H9262g of pcDNA3 1 OPT9 S pCMV SH9004 422 463 or pcDNA3 1 mu tants 1 through 8 Forty eight hours posttransfection cells were lysed on ice for 30 min in 100 H9262l of lysis buffer 50 mM Tris HCl pH 8 0 137 mM NaCl 2 mM EDTA 0 5 NP 40 10 glycerol 1 H9262g ml each of pepstatin leupeptin and pefabloc cleared of lysate 14 000 rpm for 10 min 4 C boiled at 100 C for 15 min and run on 10 sodium dodecyl sulfate polyacrylamide gel electrophoresis Invitrogen After transfer to polyvinylidene difluoride membrane Invitrogen blots were blocked in 5 milk and 0 5 bovine serum albumin BSA in phosphate buffered saline PBS washed and incubated with rabbit 419 R419D57 serum 1 100 in blocking buffer R419D57 contains polyclonal an tibodies to the first 400 amino acids of the N terminal region of the S protein as previously described 6 Blots were washed and incubated with protein G horse radish peroxidase HRP conjugate 1 4 000 Bio Rad Hercules CA Immuno fluorescence was measured with ECL plus Western Blotting Detection System Amersham Biosciences Piscataway NJ In a parallel study using immuno chemical staining 48 h posttransfection cells were washed with PBS and fixed with 100 cold methanol for 10 min at H1100220 C Fixed cells were blocked with 1 BSA in PBS washed and incubated with R419D57 serum 1 100 for1hat37 C Following primary antibody incubation cells were washed and incubated with anti rabbit immunoglobulin Ig HRP 1 2 000 Amersham Biosciences A sim ilar method was used to test mice sera immunized with mutant 3 mutant 8 OPT9 and SH9004 422 463 HEK 293T cells transfected with pcDNA3 1 OPT9 S were stained with mouse immune sera 1 50 washed and incubated with anti mouse Ig HRP 1 2 000 Amersham Biosciences Color detection was deter mined with DAB Enhanced Liquid Substrate System Sigma Aldrich working solution Animal immunizations Ten groups 2 mice per group of 6 to 8 week old female BALB c mice Charles River Laboratories were immunized with 20 H9262g of plasmid DNA in 50 H9262l saline by in vivo electroporation EP ICHOR Med System San Diego CA Mice were vaccinated every 3 weeks and sera were collected at weeks 2 and 5 Two mice immunized with mutant 3 R441A were later boosted with two additional injections of pcDNA3 1 OPT9 S at weeks 6 and 9 and sera were collected at weeks 8 and 11 An equivalent study was conducted on three groups of mice 2 mice per group immunized with pcDNA3 1 OPT9 S and R441A mutant for measurement of cytotoxic T lymphocyte responses Se rum from this study was collected at weeks 2 and 5 At the end of the study week 5 mice were euthanized and spleens were collected All animal experiments were approved by and conducted in the Laboratory Animal Research Center at the Rockefeller University Indirect enzyme linked immunosorbent assay ELISA Costar EIA RIA high binding 96 well flat bottom plates Corning Inc Corning NY were coated with 0 5 H9262g well affinity purified anti 6H11003 His tag antibodies rabbit Rockland Gil bertsville PA in coating buffer pH 9 6 0 1 M NaHCO 3 at 4 C overnight The plates were washed four times with PBST PBS containing 0 05 Tween 20 and blocked with 5 nonfat milk 0 5 BSA in PBS at 37 C for 2 h S400 and S600 proteins which have 6H11003 His tags at their C terminals 6 were then captured by anti His antibody when 200 H9262l well supernatant of 293T cells transfected with plasmids encoding proteins S400 or S600 was incubated at 37 C for 1 h After washing serial diluted 1 3 mouse immune sera was added and incubated at 37 C for 1 h After washing 1 5 000 diluted anti mouse Ig horseradish peroxi dase sheep Amersham Biosciences was incubated at 37 C for 1 h After the last wash the color was developed with substrate solution and the optical density at 450 nm was measured Values twofold above the background control were considered positive Cell mediated immune responses measured by gamma interferon ELISpot assays Cell mediated immune responses were quantified using a commercially available murine interferon ELISpot assay R Millipore Billerica MA were coated with capture antibodies Abs R Cellular Technology Ltd Cleveland OH Pseudovirus entry assay The pseudovirus was generated by cotransfecting Superfect QIAGEN 293T cells with pNL4 3Luc H11001 Env H11002 Vpr H11002 and either pcDNA3 1 OPT9 S pCMV SH9004 422 463 or pcDNA3 1 mutants 1 through 8 Cell supernatant was collected 48 h posttransfection and frozen at H11002150 C p24 was measured HIV 1 p24 Antigen EIA Beckman Coulter Fullerton CA by the Aaron Diamond AIDS Research Center Virology Core Laboratory HEK293T ACE2 or NIH 3T3 MX L SIGN cells 10 000 cells per well in 100 H9262l were plated in 96 well plates at 37 C overnight The NIH 3T3 MX L SIGN cell line was purchased from the National Institutes of Health AIDS Reagent De pository catalog no 9948 On the following day 10 ng based on p24 concen trations of pseudotype virus and 16 ng polybrene in 100 H9262l medium were added to the cells Seventy two hours after infection cells were washed with PBS and lysed 1H11003 Cell Culture Lysis Reagent Promega Madison WI Thirty microliters of cell lysate was mixed with 100 H9262l of Luciferase Assay Reagent Promega and luciferase intensity was measured Neutralization assay A pseudovirus based neutralization assay was estab lished to determine the humoral immune responses against SARS CoV 6 7 The neutralizing activity of heat inactivated sera 56 C 30 min was determined by mixing 10 ng of pseudotype virus in 30 H9262l with diluted serum in 30 H9262l at 37 C for 1 h After neutralization the mixture was combined with 16 ng poly brene in 40 H9262l medium and added to HEK293T ACE2 or 786 O cells 10 000 cells per well in 100 H9262l The 786 O cell line was purchased from the American Type Culture Collection ATCC CRL 1932 Cells were washed with PBS and lysed 1H11003 Cell Culture Lysis Reagent Promega 56 to 72 h after infection Luciferase intensity was measured and the percentage of neutralization was calculated RESULTS A deletion within the receptor binding region of the S gly coprotein abolishes immunogenicity of the major neutralizing domain and viral entry In our previous study we mapped a major neutralizing antibody determinant between residues 400 and 600 at the N terminal region of the S glycoprotein 6 This region not only serves as a dominant B cell NAb epitope but also overlaps with the ACE2 receptor binding domain RBD residues 318 to 510 34 Further analysis of this epitope revealed eight basic amino acids R426 K439 R441 R444 VOL 79 2005 MAJOR NAb AND VIRAL ENTRY DETERMINANTS OF SARS CoV 11639 on March 7 2015 by GEORGIAN COURT UNIV http jvi asm org Downloaded from H445 K447 R449 and R453 identified in a region S 420 466 of the S glycoprotein Since the positively charged resi dues correspond with the highly negatively charged surface of the cellular receptor ACE2 27 we hypothesized that electro statics may play an important role in the interaction between the S protein and ACE2 receptor To test this hypothesis we replaced the target region of 42 amino acids residues 422 to 463 with 7 neutral spacer amino acids GSGGGLE This mutant S protein called SH9004 422 463 was derived from the codon optimized full length S gene used in our parental DNA vaccine pcDNA3 1 OPT9 as previously described 6 By Western blot analysis and cell surface staining we confirmed that SH9004 422 463 is properly expressed in 293T transfected cells Protein levels were determined by immunostaining with rabbit R419D57 serum raised against the first 400 amino acids of the S protein Fig 1A and B To determine the immuno genicity of the SH9004 422 463 mutant we immunized mice with either our SH9004 422 463 mutant or pcDNA3 1 OPT9 parental plasmid DNA through in vivo electroporation EP This tech nique enhanced the generation of antibody response by more than 100 fold in comparison to intramuscular injection of na ked DNA It also gave a consistent antibody response in every vaccinated animal unpublished data Serum was collected after two injections and was measured for the ability to neu tralize our pseudovirus OPT9 6 We failed to detect the presence of NAbs after two immunizations with the SH9004 422 463 mutant H110211 10 serum dilution although binding antibod ies were observed In contrast mice immunized with pcDNA3 1 OPT9 produced substantially higher levels of NAbs 50 inhibitory concentration H110221 38 000 serum dilution Fig 1C These results indicate that the deleted region H9004 422 463 contains essential elements for inducing major NAbs To ex amine the functional determinants necessary for the deletion region and ACE2 receptor interaction the SH9004 422 463 mu tant plasmid was used to generate pseudovirus expressing lu ciferase as a reporter gene The SH9004 422 463 pseudovirus was subsequently used to infect HEK293T ACE2 cells As shown in Fig 1D SH9004 422 463 pseudovirus failed to infect cells when FIG 1 Comparison of full length S OPT9 glycoprotein versus the deletion SH9004 422 463 mutant A Western blot expression of the full length codon optimized OPT9 S glycoprotein left mutant SH9004 422 463 middle and 293T cell lysate right B 293T cell surface staining of mutant SH9004 422 463 expression left and 293T cells right Expression was measured with a polyclonal antibody to the first 400 amino acids of the S glycoprotein at a 1 100 dilution C Neutralization assay of mice sera immunized with two injections of OPT9 or the mutant SH9004 422 463 Mutant SH9004 422 463 failed to induce neutralization activity D Entry assay of the OPT9 set at 100 efficiency compared with the lack of entry with mutant SH9004 422 463 pseudotyped virus Neg negative 11640 YI ET AL J VIROL on March 7 2015 by GEORGIAN COURT UNIV http jvi asm org Downloaded from measured against the control pseudotype virus OPT9 sug gesting the loss of entry determinants Our findings also pro vide new evidence that the ACE2 receptor binding region coincides with a dominant neutralization domain in the S gly coprotein of SARS CoV Amino acid R441 determines the immunogenicity of a major neutralizing domain in the S glycoprotein Since the deletion may artificially disrupt the core structure of the receptor bind ing region we further determined the impact of individual residues on immunogenicity and viral entry Because the S 422 463 region contains a significant number of positively charged residues 9 out of 47 we sought to determine the role of these basic residues in inducing high levels of NAbs We generated eight mutants derived from pcDNA3 1 OPT9 by replacing each of the positively charged residues with alanine Fig 2A We did not mutate residue K465 because it is lo cated outside the deleted region Immunoblotting Fig 2B and immunostaining data not shown were conducted to con firm proper expression and localization of the eight mutants in 293T transfected cells Mutant plasmids 1 through 8 were sub sequently used to immunize eight groups of mice through in vivo electroporation EP After two EP immunizations 3 weeks apart we found that a single amino acid mutation R441A displayed the same phenotype as the deletion SH9004 422 463 mutant Similar to SH9004 422 463 immunizations we failed to induce detectable NAbs Fig 3 although binding antibod ies were generated Fig 4 No NAbs were detected in sera dilutions beginning at 1 10 Apparently the loss of the major neutralizing domain in R441A or SH9004 422 463 mutants re sulted in the reduced level of binding antibodies Fig 4 Using an indirect ELISA we further quantified the level of binding antibodies generated We found that R441A or SH9004 422 463 mutant was able to induce an antibody response mainly against protein S400 with a weak response to S600 at a dilution of 1 50 Conversely the R453A mutant and OPT9 induced a high level of binding antibody against protein S600 1 4 050 but not S400 H110211 50 One possible explanation is that the R441A or SH9004 422 463 mutant likely resulted in an altered conforma tional structure of the RBD which may deter the induction of neutralizing antibodies Moreover ELISpot analysis revealed a relevant cytotoxic T lymphocyte response to the R441A mu FIG 2 Construction of eight individual S glycoprotein point mutations and comparative expression A Schematic diagram of the ACE2 receptor binding region with arrows indicating the residue outside the SH9004 422 463 deletion region The eight positively charged residues are annotated H11001 above each corresponding residue and the individual mutations are shown below as a change to an alanine B Similar levels of expression of each of the mutants 1 to 8 OPT9 and cell lysate H11002 are detected in Western blots stained with a polyclonal antibody to the first 400 amino acids of the N terminus WT wild type Mut mutant FIG 3 SARS CoV specific neutralization antibody response in BALB c mice after EP DNA vaccinations Each group