【病毒外文文献】2019 Sensitive and Specific Detection of Low-Level Antibody Responses in Mild Middle East Respiratory Syndrome Coronavir

RESEARCH Middle East respiratory syndrome coronavirus MERS CoV infections in humans can cause asymptomatic to fatal lower respiratory lung disease Despite posing a probable risk for virus transmission asymptomatic to mild infections can go unnoticed a lack of seroconversion among some PCR confirmed cases has been reported We found that a MERS CoV spike S1 protein based ELISA routinely used in surveillance studies showed low sensitivity in detecting infections among PCR confirmed patients with mild clini cal symptoms and cross reactivity of human coronavirus OC43 positive serum samples Using in house S1 ELISA and protein microarray we demonstrate that most PCR confirmed MERS CoV case patients with mild infections seroconverted nonetheless some of these samples did not have detectable levels of virus neutralizing antibodies The use of a sensitive and specific serologic S1 based assay can be instrumental in the accurate estimation of MERS CoV prevalence M iddle East respiratory syndrome coronavirus MERS CoV poses a public health threat ongoing outbreaks have been reported since its detection in 2012 1 MERS CoV infection may be asymptomatic or may cause illness ranging from mild to fatal fatal infections ac count for 35 of reported cases 2 5 Dromedary camels are the virus reservoir 6 7 and pose a high risk of infecting humans in contact with them 4 7 9 These spillover events may seed outbreaks in the community 10 which occur mainly in healthcare settings 11 12 and to a lesser ex tent among patient household contacts 13 15 Although not sustained human to human transmission accounts for most reported cases 16 and may initiate outbreaks outside endemic areas as seen in the 2015 South Korea outbreak 17 However the rate of human to human transmission and total disease burden of MERS CoV are not fully clear because we lack accurate data on the frequency of asymp tomatic and mild infections Diagnostic assays with validated high sensitivity and specificity are crucial to estimate the prevalence of MERS CoV Molecular based assays have been de veloped that enable sensitive and specific diagnosis of MERS CoV infections 18 19 Although the molecular detection of viral nucleic acid by reverse transcription PCR RT PCR is the standard for MERS CoV diagnosis serologic detection remains necessary Viral nucleic acid is detectable within a limited timeframe after infection and samples from the lower respiratory tract are required for reliable results Furthermore whereas mutations in the viral regions where the PCR probes bind could lead to de creased sensitivity 20 genetically diverse MERS CoV strains may retain antigenic similarity 21 Validated se rologic assays are needed to ensure that the full spectrum of infections is identified antibodies can be detected for longer periods after infection and even if viruses mutate Several research groups and companies have developed serologic assays allowing for high throughput surveil lance for MERS CoV infections among large populations 15 19 22 25 Despite the number of serological assays devel oped none is considered to be fully validated There are 2 major challenges concerning specificity and sensitivity Sensitive and Specific Detection of Low Level Antibody Responses in Mild Middle East Respiratory Syndrome Coronavirus Infections Nisreen M A Okba V Stalin Raj Ivy Widjaja Corine H GeurtsvanKessel Erwin de Bruin Felicity D Chandler Wan Beom Park Nam Joong Kim Elmoubasher A B A Farag Mohammed Al Hajri Berend Jan Bosch Myoung don Oh Marion P G Koopmans Chantal B E M Reusken Bart L Haagmans 1868 Emerging Infectious Diseases www cdc gov eid Vol 25 No 10 October 2019 Author affiliations Erasmus Medical Center Rotterdam the Netherlands N M A Okba V S Raj C H GeurtsvanKessel E de Bruin F D Chandler M P G Koopmans C B E M Reusken B L Haagmans Utrecht University Utrecht the Netherlands I Widjaja B J Bosch Seoul National University College of Medicine Seoul South Korea W B Park N J Kim M D Oh Ministry of Public Health Doha Qatar E A B A Farag M Al Hajri DOI https doi org 10 3201 eid2509 190051Detection of Mild MERS CoV Infections aspects of MERS CoV serologic assays The first chal lenge is that 90 of the human population have anti bodies against common cold causing human corona viruses HCoVs that could cross react resulting in false positives in serologic assays especially in persons infected with viruses belonging to the same genus of coronaviruses as human seasonal coronaviruses OC43 and HKU1 26 The spike protein specifically its N ter minal S1 domain is highly immunogenic and divergent among HCoVs so it is an ideal candidate for virus spe cific serologic assays 27 The second challenge is the low antibody responses among mildly infected and as ymptomatic cases Severe MERS CoV infections result in a robust immune response allowing serologic detec tion in patients with positive or negative PCR outcomes 28 but PCR diagnosed mild or asymptomatic infec tions may cause variable immune responses that can be undetectable by serologic assays 5 15 17 Therefore a sensitive assay is necessary to avoid false negative re sults that can cause failure in detection of subclinical infections and underestimation of prevalence in serosur veillance studies We evaluated the antibody responses following severe and mild laboratory confirmed MERS CoV infections validating and comparing different as say platforms for the specific and sensitive detection of MERS CoV infections Materials and Methods Serum Samples We used a total of 292 serum samples in this study Table 1 Appendix https wwwnc cdc gov EID article 25 10 19 0051 App1 pdf The samples represented patients with serologically identified 8 and PCR confirmed MERS CoV infections 17 29 a cohort of healthy blood donors as a control group and patients confirmed by RT PCR to have non MERS CoV respiratory virus infections to as sess assay specificity The use of serum samples from the Netherlands was approved by the Erasmus Medical Center local medical ethics committee MEC approval 2014 414 The Institutional Ethics Review Board of Seoul National University Hospital approved the use of samples from pa tients in South Korea approval no 1506 093 681 The Ethics and Institutional Animal Care and Use Committees of the Medical Research Center Hamad Medical Corpo ration approved the use of samples from Qatar permit 2014 01 001 Serologic Assays We tested all serum samples for MERS CoV neutraliz ing antibodies using plaque reduction neutralization assay PRNT For S1 reactivity we used a routine ELISA rELI SA Euroimmun 15 an in house ELISA iELISA and protein microarray 8 23 For nucleocapsid reactivity we used luciferase immuno precipitation assay N LIPS 24 For S2 reactivity we used ELISA Appendix Statistical Analyses We evaluated the specificity and sensitivity and predic tive values of the assay platforms using serum samples from patients with PCR diagnosed MERS CoV infec tions respiratory virus infected patients and healthy controls We compared performance of assay platforms to PCR performance using Fisher exact test and used receiver operating characteristic ROC curve to com pare performance of different platforms We performed all statistical analyses using GraphPad Prism version 7 Results Low Antibody Responses following Mild MERS CoV Infection Several studies have proposed that antibody levels and longevity following MERS CoV infection are dependent on disease severity 5 15 17 Among PCR confirmed MERS patients mild infections may result in unde tectable or lower short lived immune responses when compared with severe infections We evaluated MERS CoV specific antibody responses in severe and mild MERS CoV infections using serum samples collected 6 9 and 12 months after disease onset from PCR confirmed MERS CoV patients from the 2015 South Korea out break 6 with severe and 5 with mild infections 17 First we tested serum samples for MERS CoV S1 antibodies using different assay platforms Figure 1 Appendix Ta ble Consistent with the earlier report 17 the routinely used rELISA detected only 2 6 mild infections Figure 1 panel A In contrast iELISA detected 5 6 mild infections Figure 1 panel B Similar results were obtained using the S1 protein microarray to screen for MERS CoV spe cific antibodies Figure 1 panel C Although these se rum samples lacked MERS CoV neutralizing antibodies 17 the presence of nucleocapsid antibodies up to 1 year postinfection in 4 6 mildly infected patients samples confirmed the results of the S1 ELISA with an assay tar geting another MERS CoV protein Figure 1 panel D All severe cases on the other hand were found positive in all tested platforms up to 1 year after disease onset indicating a robust immune response of high antibody titers in severe cases Figure 1 Appendix Table Com pared with milder infections both S1 and neutralizing an tibody responses were higher in severely infected cases confirming that antibody responses are lower following nonsevere infection Emerging Infectious Diseases www cdc gov eid Vol 25 No 10 October 2019 1869RESEARCH Specificity and Sensitivity of In house S1 ELISA and Microarray To confirm that the variation in the detection of mild cases is caused by the sensitivity of the different platforms used we further validated the platforms for specificity and sen sitivity using 292 serum samples Table 1 Using MERS CoV neutralization as the standard for MERS CoV serol ogy we tested all serum samples using plaque reduction neutralization assay PRNT 90 and for S1 S2 and nucleo capsid reactivities We assessed the specificity of the assays using serum samples from cohorts A C healthy blood donors cohort A patients with PCR confirmed acute respiratory non CoV infections cohort B and patients with acute to convales cent PCR confirmed and HCoV infections cohort C None of the serum samples from specificity cohorts A C were reactive by iELISA at the set cutoff indicating 100 specificity Figure 2 panel A Appendix We also evalu ated the sensitivity for detecting MERS CoV infections iELISA was able to detect MERS CoV infections among persons with camel contact cohort D1 who had low an tibody levels as determined by protein microarray 8 Us ing samples from acute phase PCR diagnosed patients co hort E we detected seropositivity 6 8 days postdiagnosis dpd All convalescent phase serum samples cohort F were positive up to the last time point tested 228 dpd for patient 1 and 44 dpd for patient 2 Appendix Figure 1 These results reveal the high specificity and sensitiv ity of this ELISA platform supporting our earlier findings and confirming the sensitivity of our platform in detecting low immune responses among cases of milder infection cohort G Figure 1 Overall iELISA was 100 95 CI 98 07 100 specific and 92 3 11 13 95 CI 66 7 99 6 sensitive for detection of PCR confirmed cases 96 9 overall in the tested cohorts 95 CI 84 3 99 8 Table 2 Moreover the iELISA performance was in accordance with that of the MERS CoV S1 protein microarray Figure 2 panel B S1 microarray validation showed the same pattern of specificity with no false posi tives 100 specificity 95 CI 98 07 100 in cohorts A C and was 84 6 sensitive 95 CI 57 8 97 3 for PCR confirmed cases and 93 8 overall 95 CI 79 9 98 9 Specificity of S1 as an antigen for MERS CoV serology was further supported by the rates of seropositiv ity of all the serum samples from cohorts A C 87 4 for HCoV HKU1 91 3 for HCoV OC43 96 4 for HCoV NL63 and 100 for HCoV 229E as determined by micro array Figure 2 panel C All samples were seronegative for SARS CoV and no MERS CoV false positives were detected in the iELISA and microarray Overall these re sults provided evidence for the use of S1 as a specific anti gen for MERS CoV serology We evaluated nucleocapsid and S2 antibody responses after MERS CoV infections At the set cutoff none of the control serum samples tested positive for nucleocapsid an tibodies Figure 2 panel D We detected seroconversion by nucleocapsid luciferase immunoprecipitation assay among all severely infected 4 6 66 7 mildly infected 1870 Emerging Infectious Diseases www cdc gov eid Vol 25 No 10 October 2019 Figure 1 Detection of MERS CoV specific antibody responses 6 12 months following PCR diagnosed mild and severe infections using different assays Spike S1 specific antibody responses were tested with a routinely used S1 ELISA rELISA A in house S1 ELISA iELISA B and S1 microarray C Nucleocapsid specific antibody responses were tested using a luciferase immunoprecipitation assay D Severe infections red n 5 cohort H resulted in antibody responses detected for up to 1 year by all assays while detection of mild infections green n 6 cohort G varied among assays Horizontal dotted line indicates cutoff for each assay yellow shaded area indicates serum undetected by each assay CoV coronavirus LU luminescence units MERS Middle East respiratory syndrome OD optical density Detection of Mild MERS CoV Infections and 5 18 28 asymptomatic S1 positive persons with camel contact When testing for MERS CoV S2 specific antibody responses none of the control serum samples in cohorts A C was cross reactive Figure 2 panel E where as 1 17 S1 negative samples and 1 18 S1 positive samples from persons with camel contact tested positive These findings indicate low immune responsiveness in mild MERS cases Thus when comparing the use of S1 S2 and N proteins for the detection of MERS CoV infections S1 showed the highest specificity and sensitivity among the 3 tested proteins rELISA Validation Strikingly the routinely used ELISA showed the least sen sitivity among the tested S1 platforms Table 2 Figure 1 Figure 2 panel F We saw this difference in the cohort of persons with camel contact from Qatar who had mild to asymptomatic infections and who were identified to be seropositive for MERS CoV in an earlier study 8 Fig ure 2 panel F cohort D1 Although they tested seroposi tive by iELISA and the microarray platform only 20 of those also tested positive using the rELISA platform We tested different coating conditions and found that a reduc tion in antigen coating or a loss of some conformational epitopes could have contributed to the low sensitivity seen in the rELISA versus the iELISA despite testing the same antigen S1 Figure 3 This low sensitivity confirms the likelihood of false negative detection of some MERS CoV cases using rELISA We evaluated the specificity of the rELISA platform using cohorts A C Among these serum samples from 2 patients with HCoV OC43 a CoV infection tested posi tive Figure 2 panel F but tested negative for MERS CoV neutralization by PRNT 90 and S1 antibodies by iELISA and microarray Table 3 Thus to confirm the cross reactivity of the 2 serum samples with MERS CoV S1 in rELISA we tested serum samples taken from both patients at different time points before and after OC43 infection All preinfec tion serum samples were negative but all postinfection se rum samples were positive in the rELISA Figure 4 On the contrary none of the serum samples was positive when tested by PRNT Western blot immunofluorescence assay iELISA or S1 protein microarray using commercial and in house S1 proteins indicating a false positive reaction in the rELISA Overall the rELISA was 98 97 95 CI 96 3 99 8 specific in the tested cohorts Table 3 Us ing a lower cutoff optical density ratio 0 4 to show 100 specificity and sensitivity as suggested in an earlier study 30 did increase the sensitivity from 69 2 to 84 6 but doing so reduced specificity numbers of false positive results increased from 2 to 7 and specificity decreased from 98 97 to 96 4 Appendix Figure 2 Emerging Infectious Diseases www cdc gov eid Vol 25 No 10 October 2019 1871 Table 1 Cohorts used in study of specificity and sensitivity of assays for MERS CoV Cohort Country Sample source Infection No samples Postdiagnosis range A The Netherlands Healthy blood donors negative cohort NA 50 NA B The Netherlands Non CoV respiratory infections Adenovirus 5 2 4 w Bocavirus 2 2 4 w Enterovirus 2 2 4 w HMPV 9 2 4 w Influenza A 13 2 4 w Influenza B 6 2 4 w Rhinovirus 9 2 4 w RSV 9 2 4 w PIV 1 4 2 4 w PIV 3 4 2 4 w Mycoplasma pneumoniae 1 2 4 w CMV 9 2 4 w EBV 12 2 4 w C The Netherlands Persons with recent CoV infections CoV HCoV 229E 19 2 w 1 y CoV HCoV NL63 18 2 w 1 y CoV HCoV OC43 23 2 w 1 y D1 Qatar S1 microarray positive persons with camel contact NA 19 NA D2 S1 microarray negative persons with camel contact NA 18 NA E The Netherlands RT PCR confirmed MERS case patients Acute 21 1 14 d F Convalescent 7 15 228 d G South Korea RT PCR confirmed MERS case patients Mild infection 17 6 12 mo H Severe infection 15 6 12 mo Cohorts A C were established to test assay specificity cohorts D H were established to test assay sensitivity CoV coronavirus CMV cytomegalovirus EBV Epstein Barr virus HCoV human coronavirus HMPV human metapneumovirus MERS Middle East respiratory syndrome mo month NA not applicable PIV parainfluenza virus RSV respiratory syncytial virus Cross reactivity Samples taken from 2 case patients at different time points Samples taken from 6 case patients at different time points Samples taken from 5 case patients at different time points RESEARCH Mild MERS CoV infections and Neutralizing Antibodies To investigate the difference in the neutralization respons es produced following severe and mild infections and the reliability of neutralization assays as confirmatory assays for mild infections we validated PRNT 90 for specific and sensitive detection of MERS CoV infections Although none of the healthy blood donors cohort A were reac tive the respiratory patients cohorts B and C showed low levels of cross neutralization titer 20 in 12 serum sam ples One sample with a titer of 80 Figure 2 panel G was from an HCoV OC43 patient none of the serum samples taken at 4 earlier time points from that patient showed any neutralization by PRNT data not shown All 13 serum samples tested negative for S1 antibodies in all tested plat forms Table 3 none of the serum samples was positive in 2 assays For PCR diagnosed MERS cases cohorts E H PRNT 90 showed 100 sensitivity for detecting severe cas es after the seroconversion period 14 dpd cohort F and for up to 1 year cohort H indicating strong neutralizing antibody responses In contrast results varied for mild cases cohort G Neutralizing antibodies were detected in 3 6 50 of mild 1872 Emerging Infectious Diseases www cdc gov eid Vol 25 No 10 October 2019 Figure 2 MERS CoV specific antibody responses detected by different assay platforms A In house IgG of S1 ELISA iELISA B MERS CoV S1 protein microarray C HCoV S1 microarray reactivity of non MERS CoV infected serum samples to the S1 proteins of 6 different HCoVs D nucleocapsid luciferase immunoprecipitation assay E IgG S2 ELISA F routinely used IgG S1 ELISA expressed as the ratio of optical density of sample to kit calibrator G plaque reduction neutralization test PRNT expressed as endpoint titer for 90 plaque reduction Serum samples tested were obtained from healthy blood donors n 50 cohort A patients with PCR diagnosed respiratory infections including human coronaviruses n 145 cohorts B and C S1 microarray positive n 18 cohort D1 and negative n 19 cohort D2 camel contacts and longitudinal serum samples from 2 PCR confirmed MERS CoV infected patients taken 15 228 days after diagnosis n 7 cohort F Cohort E is not included because patients in this cohort were in the acute phase of infection 14 days postdiagnosis in which seroconversion may not have occurred Cohorts A B C and F are from the Netherlands cohort D from Qatar Serum samples were tested at dilutions 1 101 for ELISA and N LIPS 1 20 for S1 microarray and 1 20 to 1 2 560 for PRNT Dotted lines indicate cutoff for each assay CoV coronavirus LU luminescence units MERS Middle East respiratory syndrome OD optical density RFU relative fluorescence units Detection of Mild MERS CoV Infections infections Appendix Table 1 highlighting lower shorter lived neutralizing respo