key: cord-320511-qnxj7d9l authors: Hueston, Linda; Kok, Jen; Guibone, Ayla; McDonald, Damien; Hone, George; Goodwin, James; Carter, Ian; Basile, Kerri; Sandaradura, Indy; Maddocks, Susan; Sintchenko, Vitali; Gilroy, Nicole; Chen, Sharon; Dwyer, Dominic E; O’Sullivan, Matthew V N title: The antibody response to SARS-CoV-2 infection date: 2020-08-27 journal: Open Forum Infect Dis DOI: 10.1093/ofid/ofaa387 sha: doc_id: 320511 cord_uid: qnxj7d9l BACKGROUND: Testing for SARS-CoV-2-specific antibodies has become an important tool, complementing nucleic acid tests (NATs) for diagnosis and for determining the prevalence of COVID-19 in population serosurveys. The magnitude and persistence of antibody responses are critical for assessing the duration of immunity. METHODS: A SARS-CoV-2-specific immunofluorescent antibody (IFA) assay for IgG, IgA and IgM was developed, and prospectively evaluated by comparison to the reference standard of NAT on respiratory tract samples from individuals with suspected COVID-19. Neutralizing antibody responses were measured in a subset of samples using a standard microneutralization assay. RESULTS: 2753 individuals were eligible for the study (126 NAT positive, prevalence 4·6%). The median ‘window period’ from illness onset to appearance of antibody was 10·2 days (range 5·8 – 14·4). The sensitivity and specificity of either SARS-CoV-2 IgG, IgA or IgM when collected 14 days or more after symptom onset was 91·3% (95% CI 84·9-95·6) and 98·9% (98·4-99·3), respectively. The negative predictive value was 99·6% (99·3-99·8). The positive predictive value of detecting any antibody class was 79·9% (73·3-85·1); this increased to 96·8% (90·7-99·0) for the combination of IgG and IgA. CONCLUSIONS: Measurement of SARS-CoV-2-specific antibody by IFA is an accurate method to diagnose COVID-19. Serological testing should be incorporated into diagnostic algorithms for SARS-CoV-2 infection to identify additional cases where NAT was not performed, and resolve cases where false-negative and false-positive NATs are suspected. The majority of individuals develop robust antibody responses following infection, but the duration of these responses and implications for immunity remain to be established. M a n u s c r i p t 3 The acute respiratory tract disease COVID-19 caused by the novel coronavirus SARS-CoV-2 emerged in Hubei province, China in December 2019. As of May 21 2020, there were more than 4.8 million cases worldwide. Diagnosis is primarily by detecting SARS-CoV-2-specific RNA by nucleic acid testing (NAT), but this has limitations, including the possibility of false-negative results due to low virus load in patients with minimal disease, inadequate respiratory tract sampling or mutations in the target sequence, and false-positive results due to contamination or nonspecific amplification. Assays for detection of SARS-CoV-2-specific antibodies in serum or plasma can be used to confirm a diagnosis of COVID-19 or to make a retrospective diagnosis in individuals who have already recovered from the acute illness and are no longer NAT positive [1] , which can be critical for outbreak investigations [2] . Such assays also permit estimates of the proportion of a population who have been infected by testing unbiased collections of sera in population-weighted serosurveys. In addition, serology assays are needed to establish the effectiveness and durability of immune responses to SARS-CoV-2 infection, for correlating humoral immune responses with disease severity [3] , for facilitating studies of convalescent plasma and hyperimmune globulin as therapeutic or prophylactic interventions [4] , and for investigating vaccine strategies. The objective of this study was to develop and evaluate an immunofluorescent antibody (IFA) test for SARS-CoV-2-specific IgG, IgM and IgA, and apply it to document the serological response in individuals with confirmed COVID-19. Since the start of the epidemic in Australia, the Public Health Laboratory Network recommended collecting acute and convalescent sera for serological assays on individuals being tested for SARS-CoV-2 infection, in addition to respiratory tract samples for NAT, though this has not been universally adopted [5] . Individuals with suspected SARS-CoV-2 infection having both respiratory A c c e p t e d M a n u s c r i p t 4 tract samples for NAT and serum samples for serological testing referred to the public health laboratory at the NSW Health Pathology-Institute for Clinical Pathology and Medical Research, Westmead from 22 th January to 6 th May 2020 were prospectively included in this study. In addition, discarded blood samples collected for routine biochemistry from patients with NAT-confirmed COVID-19 managed at Westmead Hospital were utilised as individual seroconversion panels. A specificity panel consisting of samples positive for rheumatoid factor (n=18), human influenza A virus (n=18) or Mycoplasma pneumoniae (n=8) antibodies collected during June to August 2019 were used to separately assess cross-reactivity. Detection of SARS-CoV-2 RNA was performed on respiratory tract samples and viral culture supernatant using established methods [6, 7] . SARS-CoV-2 isolated from a sample collected on 24 th January 2020 from an individual who acquired COVID-19 in Wuhan was utilised for the serological assays. The isolate belonged to SARS-CoV-2 linage A using the Phylogenetic Assignment of Named Global Outbreak Lineages Tool (Pangolin [8] ) and the consensus genome sequence has been submitted to GISAID (Accession EPI_ISL_407893 [9]). The virus was inoculated into Vero-E6 cells and examined daily for cytopathic effect (CPE) in a BSL-3 laboratory. Growth of SARS-CoV-2 was confirmed by the presence of CPE and the detection of SARS- Samples positive for SARS-CoV-2 IFA underwent SARS-CoV and MERS-CoV IFA using commercially available slides (Euroimmun, Luebeck, Germany) according to the manufacturer's instructions. Neutralizing antibody titers were determined by microneutralization using established methods [10] . Samples from individuals with NAT-confirmed SARS-CoV-2 infection that demonstrated seroconversion by IFA were used to determine the 'window period' for appearance of SARS-CoV-2- Positive reference cases were defined as persons with clinically suspected COVID-19 who had SARS-Cov-2 detected by NAT. Positive reference cases with an IFA titer of <10 beyond the observed upper range of the serologic window period were classified as having false negative serology, otherwise they were classified as true positive if an IFA titer of ≥10 was detected at initial or follow-up testing. Negative reference cases were defined as persons with suspected COVID-19 who had one or more negative SARS-CoV-2 NATs. Negative reference cases were classified as having false positive serology if an IFA titer of ≥10 was detected on initial or follow-up serology, otherwise in these cases an IFA titer of <10 was classified as true negative. Sensitivity, specificity, negative and positive predictive values and confidence intervals were calculated using medcalc (https://www.medcalc.org/). Confidence intervals for mean 'window periods' were calculated using Microsoft Excel 2013. Of 2753 individuals with suspected COVID-19 were included in the study (Figure 2 ), 1685 (61·2%) were female, and the median age was 38 years with an interquartile range of 28-51. Two thousand five hundred and seventy-seven individuals had a single serology test performed, 176 had two or more. One hundred and twenty-six individuals were SARS-CoV-2 NAT positive (prevalence 4·6%). A c c e p t e d M a n u s c r i p t 7 Fifteen individuals had appropriately timed serum collections for calculation of the serological window period. The mean window period from symptom onset to seroconversion of one or more SARS-CoV-2-specific antibody classes (IgG, IgA or IgM) was 10·2 days (95% CI 8·7-11·7) with an upper range of 14·4 days. Window periods for individual or combinations of antibody classes are shown in Table 1 . Of 126 NAT-confirmed cases, 115 had antibody from one or more classes detectable by IFA within 14 days of illness onset (sensitivity 91·3%%; 95% CI 84·9-95·6). The sensitivity and specificity, negative and positive predictive value of individual antibody classes and their combinations are shown in Table 1 . The 11 cases classified as false negative serology results were predominantly from individuals with one positive and several negative NAT tests (Supplementary Table S1 ). When the 73 cases with only a single NAT positive test were excluded, the calculated sensitivity of serology increased to 96·1% (86·5-99·5). Two individuals had multiple positive NATs but no SARS-CoV-2-specific antibody detected beyond the 14-day window period. One had severe COVID-19 and died on day 15 of illness, the second had mild disease with prolonged RNA shedding to day 26 of illness, no detectable antibody at day 17 of illness but IgA detected at a titer of 10 on day 26. The median antibody titers in each of the four-day intervals up to 28 days, followed by weekly intervals to 7 weeks, after illness onset were used to plot the dynamics of the antibody response using 425 samples from the 126 SARS-CoV-2 infected individuals (Figure 4) . The peak antibody response was seen in the third week post-illness onset, with IgA and IgM titers declining after this time point, and IgG titers declining in the sixth week after illness onset. By the seventh week, the proportion of individuals who still had detectable IgG was 84%, but only 53% for IgA and for IgM. The maximum recorded IgG titer was, on average, higher in those who were hospitalized (486, 95% CI 331-641) compared with those who were managed as an outpatient (179, 95% CI 117-242, P=0.0001). There was no correlation between antibody titers and sex, age or duration of viral RNA shedding (supplementary figures S2-S5). We show that our in-house developed IFA is a reliable diagnostic method for the detection of anti-SARS-CoV-2 antibodies. The sensitivity of this assay is greater, and window period shorter, than those reported for many other SARS-CoV-2 serology assays [1, [11] [12] [13] [14] [15] [16] [17] [18] . This may be because the antigen utilised in this IFA assay is whole virion-infected cells rather than one or two purified or recombinant viral proteins. Complementing the high sensitivity is the high specificity of our IFA, with minimal cross-reactivity against other coronaviruses other than SARS-CoV (also described in other studies [12, 16] ). The absence of reactivity in the vast majority of cases without SARS-CoV-2 infection also suggests that cross-reactivity with common endemic coronaviruses such as CoV-229E and CoV-OC43 is minimal, as has been described for SARS-CoV [19] . False-positive cases were associated with low titers of one antibody class only. The IFA assay has the advantage of being quantitative so changes in titer can be observed on paired samples which aids in interpretation of results and helps A c c e p t e d M a n u s c r i p t 10 time the onset of infection. Timing of sera collection after illness onset is important, and we recommend an acute sample in the first few days after illness onset and a second sample 14-28 days after illness onset to reliably detect seroconversion. There may be a small but important incidence of false-positive SARS-CoV-2 NAT results, which are not always easily identified [20] [21] [22] [23] [24] . This can be particularly significant in jurisdictions where the incidence of COVID-19 cases is low. We recommend serological follow-up of unexpected NAT positive cases in these circumstances in an attempt to confirm the diagnosis of COVID-19. We found that SARS-CoV-2-specific IgA provided better performance characteristics than IgM with higher sensitivity, equivalent specificity and higher titers at all time points after illness onset. This is consistent with findings in other respiratory tract infections, and has been observed previously for We chose to report any reactivity as positive in the IFA test, since the best way to resolve falsepositive results is to collect a second sample to observe a rising titer. Low antibody titers that are associated with true positive cases will generally demonstrate a rising titer on a second sample, c c e p t e d M a n u s c r i p t 11 has occurred. As with SARS-CoV, the appearance of IgG antibodies appears to be simultaneous with, or even occurs before, the appearance of IgA and IgM [13, 29] . While the association between serological response, humoral immunity and protection from reinfection remains to be established for SARS-CoV-2, it is concerning that the kinetics of the antibody responses demonstrated in this study suggest that antibody responses may be short-lived, at least when measured by IFA. Whether this translates to early susceptibility to re-infection is a priority research area. The investigations into the association of antibody response with disease severity in SARS-CoV-2, SARS-CoV and MERS-CoV infections have yielded inconsistent results. Some studies have shown early, robust antibody responses to be associated with mild disease [29, 30] , while others have indicated that severe disease is associated with higher antibody titers which may indicate a role for disease-enhancing antibodies in the pathogenesis of SARS-CoV-2 [11, [31] [32] [33] . Our study found that antibody titers were higher in individuals who were hospitalized for management of COVID-19. It is possible that certain antibody subsets are associated with protection against severe disease while others are associated with poorer outcomes [34] . Poorer immune responses may be associated with prolonged viral shedding, and the presence of antibody does not always correlate with viral clearance [35, 36] . We were unable to demonstrate associations between age, sex or duration of viral shedding and antibody titer. This study has several limitations. NAT was utilised as the reference standard for comparison to serology -false-positive and false-negative NATs may have resulted in under-estimates of the sensitivity and specificity of IFA. Antibody testing by neutralization is a more appropriate reference standard, but was only performed on a subset of samples due to the technical challenges associated with this method. Furthermore, samples classified as reference standard negative may have been collected from individuals who had an earlier, undiagnosed SARS-CoV-2 infection but who were now presenting with a second, unrelated respiratory tract or febrile illness. Such individuals may have A c c e p t e d M a n u s c r i p t 12 had negative NAT tests with persistent positive serology and would have been classified as false positive serology tests. We did not assess for cross-reactivity with other endemic coronaviruses, however, if this were to be significant we would have expected more false-positive results given that these other coronaviruses circulate commonly. The median age of participants was 38 years, with those at extremes of age being under-represented so our results may not be readily generalizable to these age groups. IFA testing is a relatively specialised technique, and while robotic instruments can be utilised for slide preparation and incubation, the reading of results is a relatively manual process requiring well trained laboratory staff. Commercially produced serology tests designed for high-throughput automated platforms such as chemiluminescent microparticle immunoassays and enzyme-linked immunosorbent assays may be more suitable than IFA for many laboratories settings, but will need to be subject to robust evaluation. Measurement of anti-SARS-CoV-2-specific antibody by IFA in serum is an accurate method for retrospective diagnosis of COVID-19. Serological testing should be selectively incorporated into diagnostic algorithms for SARS-CoV-2 infection for use in identifying additional cases where NAT was not performed, and to help resolve cases where false-negative and false-positive NATs are suspected. IFA, along with neutralizing antibody testing, will serve as appropriate comparators for other SARS-CoV-2-specific antibody assays. This includes for assessment of rapid point-of-care antibody tests, which have been shown to be less sensitive and specific [37] . Future research is awaited to further define the duration of the antibody responses to SARS-CoV-2 infection and to understand the serological correlates of protection from re-infection. Antibody assays will play a major role in the understanding of COVID-19 epidemiology, pathogenesis, immunity and immunotherapeutics. 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