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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY Nov 2005 p 1317 1321 Vol 12 No 11 1071 412X 05 08 00H110010 doi 10 1128 CDLI 12 11 1317 1321 2005 Copyright 2005 American Society for Microbiology All Rights Reserved Serological Responses in Patients with Severe Acute Respiratory Syndrome Coronavirus Infection and Cross Reactivity with Human Coronaviruses 229E OC43 and NL63 K H Chan V C C Cheng P C Y Woo S K P Lau L L M Poon Y Guan W H Seto K Y Yuen and J S M Peiris Department of Microbiology The University of Hong Kong and Queen Mary Hospital Hong Kong SAR People s Republic of China Received 17 May 2005 Returned for modification 18 July 2005 Accepted 22 August 2005 The serological response profile of severe acute respiratory syndrome SARS coronavirus CoV infection was defined by neutralization tests and subclass specific immunofluorescent IF tests using serial sera from 20 patients SARS CoV total immunoglobulin Ig IgG IgA and IgM IgGAM was the first antibody to be detectable There was no difference in time to seroconversion between the patients who survived n H11549 14 and those who died n H11549 6 Although SARS CoV IgM was still detectable by IF tests with 8 of 11 patients at 7 months postinfection the geometric mean titers dropped from 282 at 1 month postinfection to 19 at 7 months PH115490 001 In contrast neutralizing antibody and SARS CoV IgGAM and IgG antibody titers remained stable over this period The SARS CoV antibody response was sometimes associated with an increase in preexisting IF IgG antibody titers for human coronaviruses OC43 229E and NL63 There was no change in IF IgG titer for virus capsid antigen from the herpesvirus that was used as an unrelated control Epstein Barr virus In contrast patients who had OC43 infections and probably also 229E infections without prior exposure to SARS CoV had increases of antibodies specific for the infecting virus but not for SARS CoV There is a need for awareness of cross reactive antibody responses between coronaviruses when interpreting IF serology Severe acute respiratory syndrome SARS is a newly emer gent infectious disease that posed a major threat to interna tional public health in 2003 A novel coronavirus CoV was identified as the etiological agent 4 7 12 SARS CoV serol ogy by indirect immunofluorescence IF or neutralization tests is regarded as a gold standard for diagnosis of SARS coronavirus infection 12 13 In two previous studies immunoglobulin G IgG serocon version to SARS CoV occurred at a mean of 20 H11006 5 1 days 11 and 9 to 18 days 6 after onset of symptoms Follow up serum samples from some of these patients collected up to day 60 from onset of symptoms demonstrated persistently high IgG antibody titers 6 In another study IgM antibody was found to become undetectable by 11 to approximately 24 weeks after onset of illness 15 However the full serological profile re mains largely undefined Human coronaviruses 229E and OC43 have long been known to be respiratory pathogens in humans More recently NL63 and HKU 1 have been discovered as two novel corona viruses that can infect humans 17 19 The 229E and NL63 viruses are group 1 coronaviruses while OC43 and HKU 1 are classified as group 2 viruses The taxonomic classification of SARS CoV is still debated though many argue that it is a distant relative of group 2 coronaviruses 16 Previous studies had not revealed significant evidence of SARS CoV IF antibody in uninfected healthy controls 2 7 although most such individuals would be expected to have antibodies to the common human coronaviruses 229E OC43 and NL63 It has therefore been assumed that a positive SARS CoV IF result can be taken as unequivocal evidence of SARS CoV infection Conflicting opinions concerning serological cross reactions between human coronaviruses have remained When enzyme linked immunosorbent assays ELISA are used human antibody responses to 229E and OC43 can be clearly distinguished 5 14 However some cross reactive re sponses have been detected by IF 9 by complement fixation tests 8 and by ELISA using recombinant viral proteins 18 Since additional human coronaviruses have been now discov ered and in view of the global public health importance of diagnosing SARS it remains important to revisit the question of potential cross reactions in the human serological responses to coronaviruses In the present study serial sera from 20 SARS patients collected during illness and convalescence up to 6 months postinfection were tested by neutralization tests NT and by IF tests for SARS CoV specific IgG IgA IgM and total an tibody IgG IgA and IgM IgGAM Sera from patients with SARS were tested for cross reaction with other human coro naviruses 229E OC43 and NL63 in IF tests Since HKU 1 still cannot be cultured in vitro this virus was not included in the serological studies reported here Furthermore acute and convalescent phase sera from patients with 229E or OC43 in fection were tested for cross reaction with SARS CoV in im munofluorescent and neutralization tests MATERIALS AND METHODS Patients and sera Six to eight serial serum samples were collected in the first month of illness from a cohort of 20 SARS patients infected at the Amoy Corresponding author Mailing address Department of Microbi ology the University of Hong Kong University Pathology Building Queen Mary Hospital Pokfulam Rd Hong Kong SAR People s Republic of China Phone 852 2855 4888 Fax 852 2855 1241 E mail malik hkucc hku hk 1317 Gardens Hong Kong SAR Eleven of these patients had serum samples col lected at 7 months after disease onset The sera were aliquoted and stored at H1100280 C until use The 20 patients had a mean age of 39 8 years range 20 to 65 and the male to female ratio was 11 9 All patients presented with high fever and some had chills rigors myalgia malaise cough sore throat and headache Fourteen of these patients had diarrhea with the mean onset at 7 6 H11006 1 9 days Two patients were chronic hepatitis B carriers In general these patients had normal hemoglobin 13 6 H11006 1 5 g liter urea 4 4 H11006 1 4 mmol liter creatinine 87 H11006 16 H9262M liter alanine aminotransferase 42 H11006 15 U liter aspartate aminotransfer ase 47 H11006 25 U liter and white cell count 6 38 H11006 2 10 H11003 10 9 liter levels However they had low lymphocyte count 0 78H110060 24H1100310 9 liter marginally low platelet count 147 H11006 33 H11003 10 9 liter and elevated creatinine kinase 207 H11006 159 U liter levels Seven patients required admission to intensive care and five had acute respiratory distress syndrome Six patients had a fatal outcome Acute and convalescent phase sera from 11 patients with recent OC43 infec tion and 3 patients with recent 229E infection were retrieved from the serum bank of the Respiratory Pathogen research unit of the Baylor College of Med icine and kindly provided to us by R B Couch These sera had been shown to exhibit significant increases in OC43 and 229E antibody titers when tested by ELISA and microneutralization tests R B Couch personal communication Preparation of CoV infected smears SARS CoV infected Vero OC43 in fected BSC 1 and 229 infected MRC 5 cells and NL63 infected LLCMK2 cell smears were used for the study Coronavirus smears were prepared according to a method described previously 2 Briefly when 60 to 70 of cells had evidence of SARS CoV antigen expression the cells were fixed in chilled acetone for 10 min at H1100220 C and were stored at H1100280 C until use Indirect immunofluorescence assay SARS antibody detection was performed using indirect immunofluorescence Sera were screened at a dilution of 1 in 10 on infected and noninfected control cells For detection of IgG IgA or IgGAM antibodies smears were incubated for 30 min at 37 C For detection of IgM antibody sera were preabsorbed with antihuman IgG Gull sorb for 10 min at room temperature before being added onto the cells followed by incubation for 3 h at 37 C The cells were washed twice in phosphate buffered saline for 5 min each time and then anti human IgG IgA or IgM INOVA Diagnostic San Diego or IgGAM fluorescein isothiocyanate conjugates Focus Diagnostics Inc Cypress CA were added and the cells further incubated for 30 min at 37 C Sera positive at a screening dilution of 1 in 10 were titrated with serial twofold dilutions in parallel with the respective acute phase serum specimen from the same patient Sequential serum samples from each patient were assayed in the same experiment A weak SARS CoV antibody positive serum was included as a positive control in each run A positive result was scored when fluorescent intensity equaled or was higher than that of the positive control The antibody titer was taken to be the highest serum dilution giving a positive result Epstein Barr virus serology was performed according to a method described previously 1 Coronavirus neutralization test Starting with a serum dilution of 1 10 serial twofold dilutions of sera were prepared in 96 well microtiter plates Each serum dilution 0 05 ml was mixed with 0 05 ml of 200 50 tissue culture infectious doses of SARS CoV HK39849 and incubated at 37 C for 1 5 h in a CO2 incubator Then 0 1 ml of the virus serum mixture was inoculated in duplicate wells of 96 well microtiter plates with preformed monolayers of FRhK4 cells SARS CoV and further incubated at 37 C A virus back titration was per formed to assess the actual virus titer used in each experiment A cytopathic effect was observed using an inverted microscope on days 3 and 4 postinocula tion Neutralization titer was determined as the highest dilution of serum which completely suppresses the cytopathic effect in at least half of the infected wells The experiment was read when the virus back titration showed the virus dose to be 100 50 tissue culture infectious doses as expected Statistical analysis Antibody titers were transformed to log 10 and compared using the Mann Whitney U test Results Serial serum samples from patients with SARS Serial se rum samples from a cohort of 20 patients were tested by indirect immunofluorescence on cells infected with SARS CoV and human coronaviruses 229E and OC43 In addition neu tralizing antibody titers to SARS CoV were determined The mean time in days to seroconversion to SARS CoV deter mined by IF tests when using a conjugate reacting to all human immunoglobulin subclasses IgGAM was 14 2 days range 9 to 19 Durations to seroconversion in the IgG IgM and IgA class specific IF assays were 17 2 days range of 13 to 28 16 6 days range of 13 to 22 and 18 3 days range of 11 to 27 respectively The mean time to developing neutralizing anti body was 15 4 days range of 11 to 21 The mean times to seroconversion determined by the IgG IgM IgA IgGAM and neutralization test assays for patients who recovered from SARS were 17 9 16 9 19 1 14 8 and 17 3 respectively while those with fatal outcome were 17 8 16 7 19 2 14 7 and 16 9 Follow up sera at 7 months post onset of disease were avail able from 11 of these 20 patients When compared to the highest antibody titer in the first month of illness IgM antibody levels had fallen at least fourfold in five patients and were undetectable in three of them Geometric mean SARS CoV IgM titers dropped from 282 at 1 month postinfection to 19 at 7 months P H11005 0 0012 IgA antibody titers had decreased at least fourfold in five patients and remained stable in six The geometric mean IgA antibody titers at 1 and 7 months were 97 and 35 respectively P H11005 0 11 In contrast only one patient showed a fourfold or greater decrease in SARS CoV IgG antibody level the antibody level was stable in seven patients and continued to increase in three patients Total immuno globulin IgGAM titers decreased in one patient increased in two patients and remained stable in eight patients Neutraliz ing antibody titers decreased in two patients and increased in two patients and there was no significant change in seven patients The geometric mean antibody titers at 1 month and 7 months post onset of illness for IgG were 206 and 34 1 respec tively P H11005 0 31 and for IgGAM were 439 and 726 respec tively P H11005 0 49 neutralization titers remained unchanged at 124 P H11005 0 84 The sera from patients with SARS were also tested for antibody to OC43 and 229E by use of indirect immunofluores cence tests of virus infected cell smears The ranges of IgG titers against OC43 and 229E in the acute phase serum sample were 1 10 to 1 320 and H110211 10 to 1 1 280 respectively Seven SARS patients had a fourfold or greater increase in IgG titer against both OC43 and 229E Table 1 Fig 1A and B Two patients had at least fourfold rising titers only against OC43 and three had a fourfold or greater rise in antibody only against 229E One patient showed a decreased antibody level against both OC43 and 229E Seven showed no significant change in antibody level against OC43 and 229E Table 1 Fig TABLE 1 Serological cross reactivity of sera from 20 patients with SARS a with human coronavirus 229E and OC43 by immunofluorescence tests Antibody titer profile against coronavirus No of SARS patients n H11005 20 For both OC43 and 229E H11350 4 fold increase 7 For OC43 only H113504 fold increase 2 For 229E only H113504 fold increase 3 For OC43 and 229E H113504 fold decrease 1 For OC43 and 229E no significant change 7 a All 20 patients had serological and RT PCR confirmation of SARS CoV infection with an epidemiological link and clinical features compatible with SARS 1318 CHAN ET AL CLIN DIAGN LAB IMMUNOL 1C Sera from four SARS patients with a rise in IF antibody response to both OC43 and 229E and from five patients who had no cross reactive response to these viruses were tested for IF antibody responses on NL63 infected cells Five of these patients had a fourfold or greater increase in antibody titers to NL63 three of them also showed a rise in antibody titers to OC43 and 229E To determine whether these cross reactive responses were due to polyclonal activation two patients with a significant rise of antibody to SARS CoV 229E OC43 and NL63 were tested for Epstein Barr virus virus capsid antigen VCA IgG which served as an antigenically unrelated control Neither of these two patients had a significant change in Ep stein Barr virus VCA IgG titers Paired sera from patients with 229E and OC43 infections Acute and convalescent phase sera were available from 3 pa tients with 229E and 11 patients with OC43 infection There were cross reactive IF antibody responses between 229E and OC43 viruses Table 2 However all these sera remained negative by both immunofluorescent and neutralization tests for antibody to SARS CoV data not shown FIG 1 A to C Serological profile of three illustrative patients with SARS during the first month of illness The results of a comparison of antibody titers to SARS CoV by neutralization tests and by indirect immunofluorescence tests for IgGAM IgG IgM and IgA classes are shown For some patients IF antibody titers to OC43 229E and or NL63 are also shown VOL 12 2005 SEROLOGICAL RESPONSE IN PATIENTS WITH SARS 1319 Discussion Total IgGAM antibody is the antibody detectable earliest mean 14 2 days range 9 to 19 days in patients with SARS Of the subclass specific assays IgM antibodies were the earli est antibody to be detectable mean 16 6 days range 13 to 22 days Although IgM antibody titers declined significantly dur ing the first 7 months after infection we demonstrated that SARS CoV IgM remains detectable in 63 6 7 11 of patients for at least 7 months It was previously reported that IgM antibody detected by ELISA became undetectable by 11 weeks after onset of illness 15 These differences in results may be related to differences to the sensitivity of the methods used for the serology tests In contrast neutralizing antibody titers and IF IgGAM and IgG levels seem stable over the first 7 months postinfection In addition there was no significant difference in the kinetics of the appearance of the antibody responses between patients who survive or die Since serology remains the gold standard for diagnosis of SARS it is important to explore serological cross reactions between SARS CoV and the other human coronaviruses 229E and OC43 We show that 12 60 of the 20 SARS patients had fourfold rising titers to OC43 229E or both Table 1 Another recent study has shown evidence of rising antibodies to OC43 and 229E in animals immunized with SARS CoV and in human patients with SARS 3 Furthermore in the subset of patients tested some also had rising IF antibody titers to the recently discovered NL63 coronavirus Since most SARS pa tients had preexisting antibody to 229E OC43 and NL63 SARS CoV infection appears to stimulate cross reactive anti body responses to one or more of these viruses This could be due to cross reactive antigenic epitopes or to the infection resulting in polyclonal activation of antibody However pa tients who demonstrate a cross reactive coronavirus response do not have a significant change in IF antibody to a virus of an unrelated family e g Epstein Barr virus Thus the presence of cross reactive antigenic epitopes rather than polyclonal ac tivation of antibody appears to be the explanation for these findings Similarly and perhaps for similar reasons 27 of OC43 infected patients and one of three patients infected with 229E induced rising antibody titer to the other virus Table 3 Such cross reaction between human coronaviruses has been noted before in IF 9 and complement fixation tests 8 On the other hand 11 patients with recent OC43 infections and 3 with 229E infection without prior exposure to SARS CoV developed antibody to the infecting virus without induc ing a cross reacting antibody response to SARS CoV either in IF or in neutralization tests This is in agreement with the fact that there is no IF or neutralization antibody to SARS CoV detectable in uninfected individuals 2 This lack of cross reactive response to SARS CoV is possibly because these pa tients had no prior immunological memory for SARS CoV Thus there is less opportunity for a cross reactive response Given the evidence of antigenic cross reactions between coro naviruses it remains possible however that there may indeed be an increase in antibody SARS CoV titer in a patient who has previously had SARS CoV infection when the patient subsequently gets infected by OC43 or 229E This remains to be formally demonstrated However awareness of this possi bility is important from a diagnostic point of view Such cross reactions probably explain the positive results in ELISA tests based on recombinant nucleoprotein antigens 18 Further more hyperimmune animal antisera to some group 1 human TABLE 2 Cross reactivity of sera from patients with primary human coronavirus 229E or OC43 infection in immunofluorescent antibody tests Patient a Primary infection Serum status IF IgG titer for CoV IF OC43 IF 229E 1 229E Acute 40 H1102110 229E Convalescent 80 320 2 229E Acute 80 20 229E Convalescent 320 80 3 229E Acute 320 80 229E Convalescent 320 320 4 OC43 Acute 40 80 OC43 Convalescent 160 80 5 OC43 Acute 40 160 OC43 Convalescent 640 80 6 OC43 Acute 160 80 OC43 Convalescent 1 280 40 7 OC43 Acute H1102110 80 OC43 Convalescent 20 40 8 OC43 Acute 40 80 OC43 Convalescent 320 80 9 OC43 Acute 10 80 OC43 Convalescent 80 320 10 OC43 Acute 40 80 OC43 Convalescent 320 80 11 OC43 Acute 40 40 OC43 Convalescent 320 20 12 OC43 Acute H1102110 20 OC43 Convalescent 1 280 320 13 OC43 Acute H1102110 80 OC43 Convalescent 160 80 14 OC43 Acute H1102110 H1102110 OC43 Convalescent 320 20 a All patients tested negative for antibodies against SARS CoV by IF and neutralization tests TABLE 3 Cross reactive IF IgG responses to OC43 or 229E in patients with SARS Coronavirus No of patients total no showing titers rising fourfold or more against indicated CoV SARS 20 OC43 patients 11 229E patients 3 SARS CoV 20 0 0 OC43 9 11 1 229E 10 3 3 1320 CHAN ET AL CLIN DIAGN LAB IMMUNOL and animal coronaviruses was observed to cross react with SARS in vitro