Characterization of antibody response in asymptomatic and symptomatic SARS-CoV-2 infection

SARS-CoV-2 pandemic is causing high morbidity and mortality burden worldwide with unprecedented strain on health care systems. To investigate the time course of the antibody response in relation to the outcome we performed a study in hospitalized COVID-19 patients. As comparison we also investigated the time course of the antibody response in SARS-CoV-2 asymptomatic subjects. Study results show that patients produce a strong antibody response to SARS-CoV-2 with high correlation between different viral antigens (spike protein and nucleoprotein) and among antibody classes (IgA, IgG, and IgM and neutralizing antibodies). The antibody peak is reached by 3 weeks from hospital admission followed by a sharp decrease. No difference was observed in any parameter of the antibody classes, including neutralizing antibodies, between subjects who recovered or with fatal outcome. Only few asymptomatic subjects developed antibodies at detectable levels.


Introduction
On March 11, 2020, the World Health Organization (WHO) Director General declared a pandemic situation due to a novel coronavirus causing a Severe Acute Respiratory Syndrome (SARS) rapidly spreading worldwide [1].The novel coronavirus (CoV) SARS-CoV-2 has been firstly identified in Wuhan, Hubei Province, China, at the end of 2019 when a cluster of atypical pneumonia occurred [1,2].In January 2020, SARS-CoV-2 was isolated and sequenced as a CoV genetically related to the highly pathogenic CoV (SARS-CoV-1) responsible for the 2003 SARS epidemic that spread mainly in Asia with approximately 10% case fatality rate (CFR) [3].Since 2004 SARS-CoV-1 circulation in humans ended whereas a third highly pathogenic CoV emerged in 2012 in Saudi Arabia causing the Middle East Respiratory Syndrome (MERS) [2,[4][5][6][7].Since then MERS-CoV has spread to 27 countries with limited human-to-human Superiore di Sanità), Rome [29].The mean age of fatalities from COVID-19 was 80 years, 42,4% were women and more than 90% had one or more co-morbidity as ischemic heart disease, diabetes, active cancer, atrial fibrillation, dementia, and a history of stroke [31].
The emerging and rapid diffusion of COVID-19 has risen the calls for more targeted research in the field [32] helping to elucidate the mechanism of infection, protection, or rapid evolution until fatal outcome.We present here a study performed in hospitalized COVID-19 patients to investigate the time course of the antibody response in relation to the outcome, and as explorative comparison, to investigate the time course of the antibody response in SARS-CoV-2 asymptomatic subjects.

Study population
This was a retrospective study on COVID-19 patients and SARS-CoV-2 asymptomatic subjects collected between March and May 2020 during the first epidemic wave occurred in Italy.
A total of 42 COVID-19 patients, hospitalized at Humanitas Gavazzeni (Bergamo, Italy), were retrospectively selected for this study, of whom 35 (22 males and 13 females) recovered and 7 (3 males and 4 females) had a fatal outcome.All subjects were admitted to hospital with a diagnosis of interstitial pneumonia confirmed by chest radiograph or a CAT (computerized axial tomography) and had rhino-pharyngeal swab positive to SARS-CoV-2 (Real-Time PCR Thermo Fisher Scientific).Six (6) patients required care in the intensive care unit (ICU), the others were hospitalized in the general medicine unit.Out of 7 deceased patients, 3 were hospitalized in ICU and 4 in the general medicine unit.
Serum samples were collected at different time points from March to April 2020 for diagnostic/therapeutic purposes.We selected patients who had at least 5 blood samples available during the period of hospital stay (baseline, day 2, day 6, day 12-14, day 18-20, day 27-30).Demographic and clinical variables reported in this study were those collected at hospital admission.For the purpose of this study patients were categorized according to the outcome: recovered or deceased.
During the first phase of the COVID-19 epidemic, little was known about this novel CoV and there was no standard therapy, so the management changed over time.The Italian Society of Infectious and Tropical Diseases recommended as therapy hydroxychloroquine, antiviral agents, steroids, low molecular weight heparin and oxygen support in different combinations according to the clinician's evaluation.The antibiotic therapy was adopted only in case of suspected or confirmed bacterial superinfection.
Serum samples from 25 asymptomatic subjects who presented a positive rhino-pharyngeal swab for SARS-CoV-2 were collected as part of the UNICORN project and were analysed in the present study [33].
This study was approved by the Ethics Committee of the University of Siena (approval number 17373, approval date June 1, 2020), by the Ethics Committee of Humanitas Gavazzeni (approval number 236, approval date September 22, 2020 Protocol 670/20).All serum samples have been fully anonymized before testing.The UNICORN study was approved by the Ethics Committee of the University of Milan (approval number 17/20, approval date March 6, 2020).All participants signed an informed consent form.
According to the manufacturer's instructions, quantitative analysis was performed by use of a 4-parameter logistic standard curve obtained by plotting the optical density (OD) values measured for 4 calibrators against their antibody activity (U/ml) using logarithmic/linear coordinates.Antibody activities of the samples were evaluated from OD values using the generated curve and considered positive if >12 U/ml.
Virus neutralization assay.The virus neutralization (VN) assay has been performed as previously reported [20].Briefly, serum samples were heat-inactivated for 30 minutes at 56˚C and, starting from 1:10 dilution, were mixed with an equal volume of SARS-CoV-2 (2019-nCov/Italy-INMI1 strain) viral solution containing 100 Tissue Culture Infective Dose 50% (TCID50).After 1 hour of incubation at room temperature, 100μl of virus-serum mixture were added to a 96-well plate containing VERO E6 cells with 80% confluency.Plates were incubated for 3 days at 37˚C, 5% CO 2 in humidified atmosphere, then inspected for presence/ absence of cytopathic effect (CPE) by means of an inverted optical microscope.A CPE higher than 50% indicated infection.The VN titer was expressed as the reciprocal of the highest serum dilution showing protection from viral infection and CPE.

Statistical analysis
All statistical analyses were performed using Microsoft R-Open version 3.5.0(R Core Team (2018).R: A language and environment for statistical computing.R Foundation for Statistical Computing, Vienna, Austria.URL https://www.R-project.org/).For patient baseline characteristics continuous variables were evaluated using Mann-Whitney tests and for categorical variables Chi-square tests were used.Seroconversion rates were compared using Fisher's exact test.Antibody levels were statistically evaluated using t-tests.Statistical significance was set at p<0.05, two tailed.

COVID-19 patients
Between March and April 2020, a total of 42 subjects were retrospectively selected, of whom 35 recovered and 7 had a fatal outcome.The median age at admission was 64.0 years (interquartile range (IQR) 56.0-71.5)for those who recovered and 69.0 years (IQR 64.5-72.0)for deceased patients.The median length of stay in the hospital was similar in both groups with 11.0 days (IQR 9.0-24.5)and 10.0 (IQR 6.0-15.59)for recovered and deceased patients, respectively.The mean number of pre-existing conditions in recovered and deceased was 1.53 (standard deviation (SD) 1.25) and 2.0 (SD 1.41), respectively, and comorbidities were indicated in 1.88 (SD 1.36) and 3.5 (SD 3.54) of recovered and deceased patients, respectively.Forty-five per cent (45%) of patients had at least one comorbidity.Main co-morbidities were cardiovascular disease, diabetes, cerebrovascular disease, hypertension and COPD (Chronic Obstructive Pulmonary Disease).No differences were found between recovered and deceased patients when compared for symptoms at admission (fever, cough, diarrhea, dyspnea) or presence/absence of comorbidities and/or preexisting conditions.The other demographic, clinical, and blood chemistry variables collected at baseline were similar between the two groups, with exception of ALT that showed to be statistically significantly higher (p-value 0.021) in subjects who recovered (Table 1).
At hospital admission, 15 patients (35.7%) were negative for S1 IgM, 11 (26.2%) for S1 IgA, 13 (30.9%)for S1 IgG, 15 (35.7%) for NP IgG, and 10 (23.8%) for neutralizing antibodies.Five patients (11.9%) were negative to any antibody assay at the time of admission; of these, 2 died and 3 recovered.Two days after admission, 6 patients (14.3%) were still negative for S1 IgM, 7 (16.7%)for S1 IgA, 4 (9.5%) for S1 IgG, 3 (7.1%)for NP IgG, and 5 (11.9%) for neutralizing antibodies.Two patients (4.8%) were still negative to any antibody assay; of these, 1 died and 1 recovered.At 6 days of sample collection, all subjects except one (97.6%)were positive to all assays (Figs 1-5).The exception was a 40-year-old male patient, positive to S1 IgG at any time point, and borderline for NP IgG only at admission and at day 2.This patient had neutralizing antibody titers less or equal than 40 at any time point.At admission, he had fever and dyspnea with no comorbidities or preexisting conditions and recovered in 12 days.
Two patients, one recovered and one deceased both within 6 days after admission, were both negative to NP IgG at admission and at day 2. Two subjects, both recovered, were positive only for neutralizing antibodies at admission, with titers less or equal to 40.They became positive to all antibodies from day 2 onward (Figs 1-5).
Neutralizing antibodies were found in all patients, with a range from 10 to 5120.Antibody titers for patients are presented in Table 2. S1 and NP antibodies started increasing at day 2 and again at day 6.A decrease for all antibodies was observed in recovered patients at day 27-30.S1 antibody increase was similar in both recovered and deceased patients, while NP IgG titers were significantly higher in deceased patients at day 6 (p-value 0.044).At 2) in those deceased.In recovered patients, a plateau of neutralizing antibody titers was observed until day 18-20, followed by a decline at day 27-30.No significant difference was found between the two groups at any time point.However, the comparison at day 27-30 was not possible as only 2 subjects were in the deceased group (Table 2).IgM seroconversion rates were higher in the deceased at day 2 (p-value 0.043), whereas seroconversion rates for the other antibody classes were similar (Table 3).
No significant difference in antibody titers at baseline and by peak antibody level (all 5 assays combined) was found between those who survived and those deceased by using the Cox proportional hazard model.A good correlation was found among all assays as shown in Fig 6.

Asymptomatic SARS-CoV-2 infected subjects
Asymptomatic subjects were part of the UNICORN project [33] and serum samples of 25 subjects were kindly provided for the present study.Their median age was 45.0 years (IQR 36.0-60.0),8 were males and 17 females (Table 4).Twenty-one (21) subjects had the rhino-pharyngeal swab positive to SARS-CoV-2 in March 2020 and a blood draw at the same time, 19 out of the 21 subjects had a second blood draw in May, and 14 of them a third blood draw in September.Four (4) subjects had the rhino-pharyngeal swab positive in May and a blood draw at the same time, of whom 1 subject had a second blood draw in September.Asymptomatic subjects did not receive any medication during the study period that may have interfered with antibody response.Out of 25 asymptomatic subjects, 16 (64.0%)were negative to any antibody at any time point.Nine ( 9) subjects (36.0%) had at least one detectable antibody type at least at one time point.At the first time point, 6 subjects (24.0%) had positive S1 IgG, of whom 2 also had S1 IgA and NP IgG, 1 subject also had S1 IgM, S1 IgA and NP IgG, and 1 subject had S1 IgA, NP IgG and positive neutralizing antibodies.Three (3) subjects, who were negative at any antibody at the first time point, had antibodies at one of the subsequent time points.One (1) of these subjects was positive to all antibody assays at the second time point including to neutralizing antibodies.The other 2 subjects had S1 IgA, S1 IgG and NP IgG at the third time point.One subject was positive to all ELISA antibodies (S1 IgM, S1 IgA, S1 IgG, and NP IgG) at the first time point, negative at the second time point, and positive again only to S1 IgG at the third time point.Detectable neutralizing antibodies were found only in 2 subjects (8.0%): in one subject at the first and only time point available, and in the other one at the second time point, as the third time point was not available.Both subjects were positive also to S1 IgA, S1 IgG, and NP IgG, and only the first subject was positive to S1 IgM.Asymptomatic subjects with positive antibody levels in any of the assays had titers well below the level found in patients as shown in Figs 1-5 and Table 5.

Discussion
In this study we primarily evaluated the time course of the antibody response to different antigens of SARS-CoV-2 (IgG, IgM, and IgA against S1, IgG against NP, and neutralizing antibodies) in COVID-19 patients admitted to hospital for interstitial pneumonia during the first epidemic wave in March and April 2020 in Italy.No significant difference in titers was observed in any of the S1 antibody class at any time point between patients who survived and who did not survive.
The only significant difference was the higher S1 IgM seroconversion rate observed in the deceased group that may suggest an early admission to hospital after infection.In other similar studies early antibody response to S1 IgA or IgM or difference in the magnitude of the immune response to SARS-CoV-2 infection was a predictor of disease severity or progression or outcome [34][35][36][37].In this study, IgG antibody titers against NP at day 6 were significantly higher in the deceased group, as reported in other studies where an early response to NP during the first 15 days after disease onset was predictive of fatal outcome [34,36].No difference was observed for neutralizing antibodies between the recovered and deceased patients as, on the contrary, reported in other studies where neutralizing antibodies were significantly higher in patients who required ICU or died [36].One possible explanation for the similar immune  response observed in this study in both survived and deceased patients can be the fact that at admission COVID-19 patients had similar clinical and demographic characteristics.In addition, other factors may contribute to the specific immune response against SARS-CoV-2 infection that need to be considered, as the cellular-mediated immunity that may play a role in protection and disease progression.
The kinetics of the antibody response showed an increase starting from day 2 and reaching the peak between days 6 and 18-20.At day 27-30, a decline in titers was observed for any of the antibody classes.In other studies, the antibody kinetic in COVID-19 patients showed the peak at the 4 th and 5 th week after disease onset, followed by antibody decay starting at the 6 th week [37,38].This observation differs from our findings most likely due to the fact that our study period started at hospital admission when the severity of the disease was already in an advanced stage.
In this study, almost two-thirds of asymptomatic subjects were negative at any time point for any antibody class, including neutralizing antibodies.Among the few subjects with detectable antibodies, all were positive to S1 IgG, and, none of these subjects was positive to any of the other antibody classes if not positive to S1 IgG.This is difficult to explain.It may be due to the fact that the S protein is deemed the immunodominant antigen of SARS-CoV-2.In accordance with a similar study, antibody titres in asymptomatic subjects were sensibly lower as compared to COVID-19 patients [39].It has been reported that asymptomatic subjects have a low viral load in the nasopharynges as assessed by RT-PCR, and most likely a lack or a defective viral replication that induces a weak or any antibody response [40].Although memory B and T cells are likely to be primed in SARS-CoV-2 infected subjects with undetectable antibodies, the question of whether they should be vaccinated is critical now that effective vaccines are available against COVID-19.In this study, a good correlation between S1 and NP protein-based ELISA and the VN assay was observed in COVID-19 patients, although less evident in asymptomatic subjects since only 2 of them had detectable neutralizing antibodies in addition to other ELISA antibody classes.It is acknowledged that antibodies with neutralizing activity should retain enough avidity or have a sufficient concentration or both [41,42].In fact, ELISA detects antibody against individual antigens that may not retain neutralizing properties if not in quantity.This may explain the difference in the correlation between ELISA-based assays and neutralizing antibodies in COVID-19 patients and asymptomatic subjects observed in this study.
This study has some limitations.The sample size was small due to the availability of subjects with severe COVID-19 disease who had a sufficient number of blood samples for measuring the time course of antibody for at least one month.This may introduce a bias.However, the ratio between deceased and recovered patients (7 out of 42, 16,6%) falls in the acceptable range (from 5.7% to 30.4%) described in the literature [18,43].The retrospective nature of the study and the collection of COVID-19 samples carried out in a single center introduce some limitations.The findings from this study do not allow to predict the kinetics of the antibody decay over time in patients who recovered from COVID-19, and who may be susceptible to reinfection over time, since no follow-up samples after discharge were available.In conclusion, the results of this study show that COVID-19 patients produce a strong antibody response to SARS-CoV-2 with high correlation between different viral antigens (S1 and NP) and among antibody classes (IgA, IgG, and IgM and neutralizing antibodies).The peak is reached by three weeks from hospital admission followed by a sharp decrease.On the contrary, only few asymptomatic subjects develop antibodies at detectable levels, and significantly lower compared to COVID-19 patients.Currently, no correlates of protection are established for COVID-19.As cases of reinfection are reported [44][45][46][47] and since neutralizing antibodies are rarely produced in asymptomatic subjects, the findings of this study support the current recommendation to vaccinate subjects with a previous SARS-CoV-2 infection as well as those who recovered from COVID-1.