【病毒外文文献】2007 Recombinant Protein-Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike

CLINICAL AND VACCINE IMMUNOLOGY Mar 2007 p 331 333 Vol 14 No 3 1556 6811 07 08 00H110010 doi 10 1128 CVI 00351 06 Copyright 2007 American Society for Microbiology All Rights Reserved Recombinant Protein Based Assays for Detection of Antibodies to Severe Acute Respiratory Syndrome Coronavirus Spike and Nucleocapsid Proteins H17188 Lia M Haynes 1 Congrong Miao 1 Jennifer L Harcourt 1 Joel M Montgomery 2 Mai Quynh Le 7 Sergey A Dryga 5 Kurt I Kamrud 5 Bryan Rivers 5 Gregory J Babcock 6 Jennifer Betts Oliver 2 James A Comer 2 Mary Reynolds 3 Timothy M Uyeki 4 Daniel Bausch 2 Thomas Ksiazek 2 William Thomas 6 Harold Alterson 5 Jonathan Smith 5 Donna M Ambrosino 6 and Larry J Anderson 1 National Centers for Immunization and Respiratory Diseases Division of Viral Diseases Respiratory and Gastroenteritis Viruses Branch 1 Division of Viral and Rickettsial Diseases Special Pathogens Branch 2 Poxvirus Activity 3 and Influenza Division 4 Centers for Disease Control and Prevention CDC 1600 Clifton Rd NE Atlanta Georgia AlphaVax Inc Research Triangle Park North Carolina 5 Massachusetts Biologic Laboratories University of Massachusetts Medical School Jamaica Plain Massachusetts 6 and National Institute of Hygiene and Epidemiology Hanoi Vietnam 7 Received 26 September 2006 Returned for modification 31 October 2006 Accepted 8 January 2007 Recombinant severe acute respiratory syndrome SARS nucleocapsid and spike protein based immuno globulin G immunoassays were developed and evaluated Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies The 2003 outbreak of severe acute respiratory syndrome SARS lasted fewer than 9 months yet it had a major impact on public health and socioeconomics Since the end of the SARS outbreak there have been 17 identified SARS associ ated coronavirus SARS CoV infections 6 from direct labo ratory exposure 1 of which resulted in community transmission to seven individuals and 4 sporadic community acquired in fections 9 Since additional cases may occur and could if undetected quickly lead to another global outbreak it is im portant to continue to improve our ability to reliably monitor SARS CoV infections 3 16 18 As with other coronaviruses the spike S and nucleocapsid N proteins are abundantly expressed during virus infection and are most effective among the coronavirus structural pro teins at inducing antibody responses 10 14 15 23 Previous studies have demonstrated the utility of anti N and anti S pro teins in the diagnosis of SARS CoV infections 2 5 12 21 In this study we describe the evaluation and comparison of re combinant spike and nucleocapsid enzyme linked immunosor bent assays ELISAs for specifically detecting SARS CoV in fection The recombinant full length SARS N gene was amplified from SARS CoV RNA Urbani strain modified to contain a C terminal His 6 tag and cloned into the Venezuelan equine encephalitis virus replicon vector 17 Baby hamster kidney BHK cells were transfected with SARS N replicon RNA by electroporation Cells were harvested and expressed protein was purified by metal affinity chromatography and analyzed by silver staining and Western blot analysis for the appropriately sized 50 kDa immunoreactive protein 8 The control anti gen the nontoxic 50 kDa C terminal fragment of the botu linum neurotoxin serotype A BoNt HcA was expressed as described above 7 The soluble codon optimized SARS CoV S glycoprotein 170 kDa amino acids 1 to 1190 S1190 and the control antigen truncated angiotensin converting enzyme 2 120 kDa glycosylated tACE 2 were cloned into pcDNA3 1 Myc His and expressed in HEK 293T 17 cells The proteins were puri fied using metal affinity chromatography and analyzed as de scribed by Babcock et al 1 Recombinant SARS N and S protein indirect ELISAs were developed using a modified version of the inactivated whole virus ELISA described by Ksiazek et al 6 Briefly ELISA plates Immulon were coated with either purified recombi nant N protein 12 5 ng well and the control antigen BoNT HcA or purified His 6 c myc tagged recombinant S1190 protein 12 5 ng well and the control antigen tACE 2 The plates were washed and incubated with serum diluted 1 400 in phosphate buffered saline PBS containing skim milk and Tween 20 PBS T M for1hat37 C and washed and incubated again with horseradish peroxidase conjugated goat anti human im munoglobulin G IgG 1 4 000 heavy plus light chain KPL in PBS T M After washing substrate ABTS 2 2H11032 azi nobis 3 ethylbenzthiazolinesulfonic acid was added and the absorbance read at 405 nm with a 490 nm reference filter ELISA conditions were optimized using antibody positive and negative serum specimens and the resultant assays were then evaluated using available serum samples collected from 61 patients from Vietnam and Taiwan with laboratory con firmed SARS CoV infection 2 to 150 days postonset of symp Corresponding author Mailing address Centers for Disease Control and Prevention 1600 Clifton Rd NE Mailstop G 18 At lanta GA 30333 Phone 404 639 4004 Fax 404 639 4005 E mail loh5 cdc gov H17188 Published ahead of print on 17 January 2007 331 on March 8 2015 by DAHLGREN MEDICAL LIBRARY http cvi asm org Downloaded from toms dpo from 46 U S patients with non SARS related respiratory infections and from 483 healthy U S donors with no exposure to SARS CoV An additional 10 serum samples were collected from non SARS patients from Vietnam and Taiwan Specimens used in this study were exempt from CDC Institutional Review Board review under 45 CFR 46 101 b 4 20 ELISA results from representative serum specimens from patients with SARS and healthy controls are shown in Fig 1 The sums of the mean absorbance values for the healthy con trols and three times the standard deviations for the S and N protein assays were 0 201 and 0 210 respectively With these cutoff values 7 of the 483 serum samples from healthy donors had absorbance values above the cutoff value in the N protein ELISA and 3 samples were above the cutoff for the S protein ELISA resulting in specificities of 98 6 and 99 4 for the N and S protein ELISAs respectively Table 1 Control samples reactive to either the N or S protein did not show reactivity to the alternate protein and also showed no reactivity against the inactivated SARS CoV lysate by ELISA In these samples reactivity may have been due to nonspecific reactivity or cross reacting antibodies To further evaluate the specificity of the assays available sera from 46 individuals infected with non SARS related res piratory viruses including human coronavirus strains 229E HCoV 229E n H11005 22 paired specimens and HCoV OC43 n H11005 17 paired specimens respiratory syncytial virus n H11005 2 human parainfluenza virus 2 n H11005 1 and 3 n H11005 1 influenza B virus n H11005 1 adenovirus n H11005 1 and mumps virus n H11005 1 were analyzed None of the serum samples were positive by either assay data not shown In addition serum samples from non SARS patients from Taiwan and Vietnam showed no re activity by either assay data not shown All 61 sera from SARS cases were antibody positive to the N protein and 59 were positive to the S protein Two acute phase specimens H1134920 dpo were weakly reactive to the S protein and fell below the assay cutoff of 0 201 All were also positive by both immunofluorescence antibody testing and ELISA using whole gamma irradiated SARS CoV as the antigen data not shown The sensitivities for the N and S protein assays were 100 and 96 7 respectively Persistent levels of SARS CoV IgG have been detected in SARS cases for several months and up to 2 years after disease onset 4 11 12 19 21 In this study serum samples from 48 SARS patients from Vietnam and the United States were col lected 221 to 735 days 44 from days 221 to 250 4 from days 633 to 735 after the onset of illness and tested for the presence of SARS CoV and N and S protein specific IgG by ELISA Antibodies IgG specific to whole virus N protein and S protein were detected in 40 83 3 45 93 8 and 36 75 of the samples tested respectively Interestingly anti SARS CoV and S antibodies were detected in three patients two of whom also demonstrated a response to the N protein almost 2 years postonset of symptoms SARS titers H11005 1 400 to 1 1 600 n H11005 3 N protein titer H11005 1 400 n H11005 2 S protein titer H11005 1 400 to 1 1 600 n H11005 3 Our evaluation of these ELISAs illustrates the value of hav ing several assay systems to detect and then confirm a SARS CoV infection Although very few serum specimens from un exposed persons H110211 5 tested positive for SARS CoV infection the potential for cross reactivity between SARS CoV and other coronaviruses including the known human coronaviruses HCoV OC43 HCoV 229E and recently identi fied HKU1 and NL63 remains a concern 12 13 22 Whether these positive results are due to nonspecific reactivity to the recombinant SARS N protein or to cross reactivity to other human CoVs requires further study The use of protein frag ments or peptides instead of the whole recombinant N pro tein for antibody detection may resolve the issue of potential cross reactivity with proteins of other human CoVs and is the focus of further study These false positives could present a public health dilemma as illustrated in the laboratory evalua tion of four sporadic cases reported by Liang et al 9 In those cases it was necessary to conduct confirmatory testing using several different types of assays because there was concern FIG 1 Scatter chart of absorbance values with representative sera from both SARS patients and healthy controls for IgG antibodies to recombinant spike protein A and recombinant nucleocapsid protein B Specimens were obtained from 61 SARS patients at 2 to 150 days postonset of symptoms and from 384 healthy U S blood donors Results are plotted as A 405 values with a 490 nm reference filter The black line indicates the cutoff values of 0 201 A and 0 210 B for each assay TABLE 1 Comparison of sensitivities and specificities of recombinant nucleocapsid protein and spike protein based ELISAs Antigen SARS patients Healthy donors No with positive reactivity total Sensitivity No with negative reactivity total Specificity SARS N 61 61 100 476 483 98 5 SARS S 59 61 96 7 480 483 99 4 332 NOTES CLIN VACCINE IMMUNOL on March 8 2015 by DAHLGREN MEDICAL LIBRARY http cvi asm org Downloaded from that infection with non SARS coronaviruses may induce cross reacting antibodies 9 Our data from that study suggest that a combination of assays may be needed to confirm the speci ficity of presumed SARS antibodies Since the costs i e public health interventions or outbreak response of detecting a false positive result and not detecting a case of SARS CoV infection are both high it is important to have well characterized detec tion and confirmatory assays In the absence of virus specific control measures e g a vaccine or antiviral drug the key to controlling a reemergence of SARS is rapid diagnosis and implementation of infection control measures i e isolating cases and identifying and managing contacts to prevent further transmission The development of well characterized detection and confirmatory serologic tests is the key to laboratory diag nostic support should SARS reemerge These two ELISAs can be used as components of the SARS diagnostic system These assays can also be used to study the kinetics of the protein specific SARS antibody response and to help characterize SARS immunity and the pathogenesis of disease We thank Der Yuan Wang Bureau of Food and Drug Analysis Department of Health Taiwan Republic of China Mei ying W Yu CBER Food and Drug Administration Bethesda MD and Li Ching Hsu Center for Disease Control Department of Health Taiwan Republic of China for providing a panel of sera from SARS patients Thanks also to Ann Falsey University of Rochester School of Medi cine Rochester NY and Dean Erdman CDC Atlanta GA for providing sera from patients with non SARS related respiratory infec tions and Debi Cannon CDC Atlanta GA for technical assistance The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the CDC REFERENCES 1 Babcock G J D J Esshaki W D Thomas Jr and D M Ambrosino 2004 Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor J Virol 78 4552 4560 2 Chang M S Y T Lu S T Ho C C Wu T Y Wei C J Chen Y T Hsu P C Chu C H Chen J M Chu Y L Jan C C Hung C C Fan and Y C Yang 2004 Antibody detection of SARS CoV spike and nucleocapsid pro tein Biochem Biophys Res Commun 314 931 936 3 Drosten C S Gunther W Preiser S van der Werf H R Brodt S Becker H Rabenau M Panning L Kolesnikova R A Fouchier A Berger A M Burguiere J Cinatl M Eickmann N Escriou K 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