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Title: Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019 Brief Title: Antibody responses in COVID-19 patients Summary Background
Abstract
The novel coronavirus SARS-CoV-2 is a newly emerging virus. The antibody response in infected patient remains largely unknown, and the clinical values of antibody testing have not been fully demonstrated.
A total of 173 patients with confirmed SARS-CoV-2 infection were enrolled. Their serial plasma samples (n = 535) collected during the hospitalization period were tested for total antibodies (Ab), IgM and IgG against SARS-CoV-2 using immunoassays. The dynamics of antibodies with the progress and severity of disease was analyzed.
Among 173 patients, the seroconversion rate for Ab, IgM and IgG was 93.1% (161/173), 82.7% (143/173) and 64.7% (112/173), respectively. Twelve patients who had not seroconverted were those only blood samples at the early stage of illness were collected.
The seroconversion sequentially appeared for Ab, IgM and then IgG, with a median time of 11, 12 and 14 days, respectively. The presence of antibodies was < 40% among patients in the first 7 days of illness, and then rapidly increased to 100.0%, 94.3% and 79.8% for Ab, IgM and IgG respectively since day 15 after onset. In contrast, the positive rate of RNA decreased from 66.7% (58/87) in samples collected before day 7 to 45.5% (25/55) during days 15 to 39. Combining RNA and antibody detections significantly improved the sensitivity of pathogenic diagnosis for COVID-19 patients (p < 0.001), even in early phase of 1-week since onset (p = 0.007). Moreover, a higher titer of Ab was independently associated with a worse clinical classification (p = 0.006).
The antibody detection offers vital clinical information during the course of SARS-CoV-2 infection. The findings provide strong empirical support for the routine application of serological testing in the diagnosis and management of COVID-19 patients.
Since early December of 2019 and up to February 24, 2020, over 79, 000 cases of coronavirus disease 2019 (COVID-19) caused by novel coronavirus (SARS-CoV-2) infection, with over 2, 600 death cases infection have been reported in 26 countries with a majority of occurrences in China. 1 The World Health Organization declared a public health emergency of international concern on January 30, 2020. Public health authorities around the world react promptly to prevent further spread of the virus.
According to recent reports, most of COVID-19 patients have an incubation period of 3 to 7 days. 2 Fever, cough and fatigue are the most common symptoms, whereas nasal congestion, runny and diarrhea are only noted in a small part of the patients. 3 Severe cases might rapidly progress to acute respiratory distress syndrome (ARDS), septic shock and difficult-to-tackle metabolic acidosis and bleeding and coagulation dysfunction. 4 It should be noted that some of COVID-19 patients only had mild atypical symptoms initially, even for severe and critical cases. 5 The chest computed tomography features of COVID-19 patients were characterized by the ground-glass opacity and bilateral patchy shadowing. 6 For laboratory test, it was reported that over 80% of patients had lymphopenia, and most of patients had elevated C-reactive protein. 7 However, the above-mentioned clinical and laboratory characteristics are not easily distinguishable from pneumonia induced by infection with other common respiratory tract pathogens such as influenza virus, streptococcus pneumoniae and mycoplasma pneumoniae.
The timely and accurate diagnosis of the SARS-CoV-2 infection is the cornerstone of All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint the efforts to provide appropriated treatment for patients, to limit further spread of the virus and ultimately to eliminate the virus from human society. Currently, viral RNA detection by several polymerase chain reaction (PCR) based technics is almost the only way to confirm the diagnosis of SARS-CoV-2 infection in practice. On the other hand, RNA testing based on throat or nasopharyngeal swabs brought out negligible false-negative risk. 8 The reported positive rate varied for different swab specimens in COVID-19 patients. 3, 9 Many cases that were strongly epidemiologically linked to SARS-CoV-2 exposure and with typical lung radiological findings remained RNA negative in their upper respiratory tract samples. There are four potential reasons: 1) the viral loads in upper respiratory tract samples are much lower than that in lower respiratory tract samples in COVID-19 patients; 9 2) the releasing viral loads of patients in different stage of infection varies with a wide range; 10 3) the collection of high-quality swab specimen requires skillful health-workers; and 4) PCR reagents from different sources have high variance. Consequently, these problems lead to a noteworthy delay of early diagnosis and following management and propose serious challenge to providing timely life support treatment and preventive quarantine.
For many known pathogenic viruses, it has been a routine practice to make a diagnosis of acute infection according to the serological findings for a long time.
Comparing to PCR, serological testing is advantageous with faster turn-around time, high-throughput and less workload. However, the clinical value of antibodies largely depends on the understanding of host antibody responses during infection. Given that SARS-CoV-2 is a newly emerging virus, the antibody response in COVID-19 patients All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint 6 remains largely unknown. To mitigate this knowledge gap, and to provide scientific analysis on the benefit of antibody testing when used in combination with the current RNA testing, this study investigates the dynamics of total antibody (Ab), IgM and IgG antibody against SARS-CoV-2 in serial blood samples collected from 173 confirmed COVID-19 patients and provides discussion on the clinical value of antibody testing. Written informed consent was obtained from each enrolled patient.
The total antibody (Ab), IgM antibody and IgG antibody against SARS-CoV-2 in plasma samples were tested using enzyme linked immunosorbent assay (ELISA) kits supplied All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is
For continue variables description, mean with standard deviation was used for normal distribution data and median with interquartile range (IQR) was used for non-normal distribution data. Cumulative seroconversion rates were calculated by Kaplan-Meier method. The association between antibody level and severity of illness were estimated by generalized estimating equations (GEE) model with logit link function. All statistical analysis was conducted by SAS 9.4 (SAS Institute, Cary, NC, USA).
The funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint
Among totally 368 cases of COVID-19 patients admitted in the hospital (before Feb 9, 2020), 173 patients of them (47%) were enrolled in the study ( Table 1) (Table 1) . Among them, 32 (18.5%) were in critical illness condition with ARDS or oxygen saturation < 93% who required mechanical ventilation either invasively or non-invasively, and the remaining 141 (81.5%) had mild to moderate syndromes were in non-critical condition. By February 19, a total of 62 patients (35.8%, 54 were in non-critical group and 8 were in critical group) were recovered and discharged from hospital and 2 (1.1%, both were in critical group) patients died with underlying chronic disease.
A total of 535 plasma samples collected during the hospitalization period of the 173 patients were tested for antibodies against SARS-CoV-2. The seroconversion rate for Ab, IgM and IgG was 93.1% (161/173), 82.7% (143/173) and 64.7% (112/173), respectively (Table 1) . Twelve patients who remained seronegative for Ab testing possibly due to that their samples involved were all collected at the early stage of All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint illness (10 earlier than day 10, the other two on day 11 and 13 after onset). The cumulative seroconversion curve showed that the rate for Ab and IgM reached 100% around 1 month of illness day. The seroconversion was sequential appeared for Ab, IgM and then IgG ( Figure 1A ). The median time to Ab, IgM and IgG seroconversion was 11, 12 and 14 days, separately. One of two patients tested on the onset day was seropositive. Overall, the seroconversion of Ab was significantly quicker than that of IgM (p = 0.012) and IgG (p < 0.001), that possibly attributed to the double-antigen sandwich form of the assay used which usually show much higher sensitivity than capture assay (IgM) and indirect assay (IgG). Moreover, all isotypes of antibodies against viral antigen, including IgM, IgA and IgG, can be detected by double-sandwich based assay, which may also contribute to the superior performance of Ab test. In comparisons of seroconversion rates of antibodies between critical and non-critical patients, none of the three markers showed significant difference ( Figure 1B , 1C and ID, p > 0.05).
We analyzed the detectability of RNA test and antibody assays according to the time course since illness onset in the cohort. As the results shown in Figure 2 and Table 2, in the early phase of illness within 7-day since onset, the RNA test had the highest sensitivity of 66.7%, whereas the antibody assays only presented a positive rate of 38.3%. However, the sensitivity of Ab overtook that of RNA test since day 8 after onset and reached over 90% across day 12 after onset ( Figure 2 ). In samples from patients during day 8-14 after onset, the sensitivities of Ab (89.6%), IgM (73.3%) and IgG All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is (56/57) and 100% (30/30) had detectable antibody in total Ab assay, respectively (Table 3) . Whatever, combined use of the tests of RNA and Ab improved markedly the sensitivities of pathogenic-diagnosis for COVID-19 patients in different phases ( Table 2 , Ab+RNA).
To investigate the dynamics of antibody level according disease course, the antibody levels were expressed using the relative binding signals compared to the cutoff value of each assay (S/CO). The longitudinal changes of antibody and RNA in 9
representative patients, including 6 in non-critical group ( Figure 3A ) and 3 in critical group ( Figure 3B ), were presented in Figure 3 . The first positive time point of RNA tests appeared earlier than that of Ab in 7 of 9 patients, except for the case 185 (Ab was detectable 2 days earlier than RNA) and the case 111 (at the same day). It should be noted that the risings of antibodies were not always accompanied by RNA clearance, particularly in the 3 critical patients. This finding suggested that antibodies may not be sufficient to clear the virus. In the pooled analyses on all involved patients, the average antibody levels showed a marked increase since about 1-week after onset and All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint continuously elevated during the next 2 weeks ( Figure 4A ). Further analyses suggested that there was no significant difference on the average S/CO value of Ab tests between critical and non-critical patients before day 12 after onset ( Figure 4B ).
However, critical patients showed significantly higher Ab S/CO values than non-critical cases in about 2-week after onset (p = 0.02) and this association was not significant in either IgM or IgG tests (data not shown). For further exploration, we determined the relative Ab titer of these samples (expressed as relative optical density, rOD) by serial dilution measurements of each sample. The quantitative data of Ab titers also revealed a significant difference (p = 0.004) between patients in critical and non-critical groups ( Figure 4C ). Multivariate longitudinal GEE analyses suggested that age (β = 0.139, p < 0.001), gender (β = 1.415, p = 0.006) and Ab titer (β = 0.336, p = 0.006) were the independent factors strongly associated with the clinical classification based on the severity (Table 4) .
The present data demonstrated that typical antibody responses to acute viral infection are wildly induced in COVID-19 patients. All patients were seroconverted, except for 12 patients with only samples collected at the early stage of illness (before 13 day of onset). To be expected, the total antibody was first detected, followed by IgM and IgG.
The seroconversion rate and the antibody levels increased rapidly during the first two weeks, the cumulative seropositive rate reached 50% on the 11th day and 100% on the 39th day. The seroconversion time of total antibody, IgM and IgG antibodies appeared consequently (p < 0.05) with a median seroconversion day of 11, 12 and 14, All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is Thus, the antibody testing might play vital roles in the following settings: 1) for the suspected patient under the initial visit or clinically diagnosed patients has not been All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint 13 confirmed by RNA testing, the positive result of antibody increases the confidence to make a COVID-19 diagnosis; 2) for healthy close contact who is in the quarantine period, he/she should be deemed as a probable carriers if antibody positive, then the RNA should be tested more frequently and the close contacts of him/her should be observed; 3) for the RNA confirmed patient, seropositive indicates that the specific immune response had been induced. Besides, epidemiological studies could be conducted using immunoassays. Additional, it can play an important role in searching potential animal hosts for SARS-CoV-2 using Ab-ELISA because the double antigen sandwich method makes it free from species restriction. It has been less than three months since the SARS-CoV-2 virus first invaded human society, and the prevalence of antibody against SARS-CoV-2 is nearly zero. Therefore, at least during the current outbreak which is likely to continue to May or June 2020, individuals who are seropositive could be a probably preceding infector of SARS-CoV-2. During this short period, therefore, the total antibody could be considered as a recent infection marker similar as IgM antibody. Our data showed that the sensitivity of total antibody testing is higher than IgM or IgG. Therefore, the total antibody detection should be given high priority to be implemented in current clinical and public health practice. If, unfortunately, SARS-CoV-2 become a common respiratory transmission pathogen lasting in human society, such as influenza viruses or low-pathogenicity types of coronaviruses, rather than be completely eradicated as its relative SARS-CoV-1 virus, the serological diagnosis of acute SARS-CoV-2 infection will more depend on the detection of IgM antibody in post-epidemic areas, such as Wuhan, China, in the subsequent epidemic All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint 14 seasons. The total antibody and IgG antibody could be used to understand the epidemiology of SARS-CoV-2 infection and to assist in determining the level of humoral immune response in patients. Even then, the total antibody will be a more sensitive marker for sentinel monitoring of imported cases in naive community.
In addition to the diagnosis value of Ab test, our study revealed a strong positive correlation between clinical severity and antibody titer since 2-week after illness onset, for the first time in COVID-19 patients. The results suggested that a high titer of total antibodies against the virus may be considered as a risk factor of critical illness, independently from older age, male gender and comorbidities (Table 4) It should be noted that there were some limitations of this study. First, for most of RNA tests of the patients were based on upper respiratory tract specimens, the positive rate may be higher in detection using lower respiratory tract specimens, such as bronchoalveolar lavage fluid, deep tracheal aspirates, and induced sputum may yield higher sensitivity for RNA tests. Second, we cannot evaluate the persistence of antibodies because samples were collected during the acute illness course of patients. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint 15 Third, although it had shown good specificity in healthy people, the cross-reactivity among the different coronaviruses cannot be accurately assessed because we cannot obtain blood samples from SARS-CoV-1 and other coronaviruses infection patients.
Future studies are needed to a better understanding of the antibody response profile of SARS-CoV-2 infection.
In conclusion, the findings demonstrate that antibody tests have important diagnosis value in addition to RNA tests. These findings provide strong evidence for the routine application of serological antibody assays in the diagnosis and clinical management of COVID-19 patients.
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The copyright holder for this preprint (which was not peer-reviewed) is
We declare no competing interests. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is All rights reserved. No reuse allowed without permission.
the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.03.02.20030189 doi: medRxiv preprint * RNA was tested using throat/nasal swab sample.
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the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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