key: cord-0982161-2dl3p2do
authors: den Hartog, Gerco; Vos, Eric R A; van den Hoogen, Lotus L; van Boven, Michiel; Schepp, Rutger M; Smits, Gaby; van Vliet, Jeffrey; Woudstra, Linde; Wijmenga-Monsuur, Alienke J; van Hagen, Cheyenne C E; Sanders, Elisabeth A M; de Melker, Hester E; van der Klis, Fiona R M; van Binnendijk, Robert S
title: Persistence of antibodies to SARS-CoV-2 in relation to symptoms in a nationwide prospective study
date: 2021-02-24
journal: Clin Infect Dis
DOI: 10.1093/cid/ciab172
sha: fcd9487d4b2a296e2967cea9df86d83c86ef5dde
doc_id: 982161
cord_uid: 2dl3p2do

BACKGROUND: Assessing the duration of immunity following infection with SARS-CoV-2 is a first priority to gauge the degree of protection following infection. Such knowledge is lacking especially in the general population. Here, we studied changes in Immunoglobulin (Ig) isotype seropositivity and IgG binding strength of SARS-CoV-2-specific serum antibodies up to 7 months following onset of symptoms in a nationwide sample. METHODS: Participants from a prospective representative serological study in the Netherlands were included based on IgG seroconversion to the Spike S1 protein of SARS-CoV-2 (N=353), with up to three consecutive serum samples per seroconverted participant (N=738). IgM, IgA and IgG antibody concentrations to S1, and increase in IgG avidity in relation to time since onset of disease symptoms, were determined. RESULTS: While SARS-CoV-2-specific IgM and IgA antibodies declined rapidly after the first month post onset of disease, specific IgG was still present in 92% (95% confidence interval, CI, 89-95) of the participants after 7 months. The estimated 2-fold decrease of IgG antibodies was 158 days (95% CI 136-189). Concentrations sustained better in persons reporting significant symptoms compared to asymptomatic persons or those with mild upper respiratory complaints only. Similarly, avidity of IgG antibodies for symptomatic persons showed a steeper increase over time compared with persons with mild or no symptoms (p=0.022). CONCLUSION: SARS-CoV-2-specific IgG antibodies persist and show increasing avidity over time, indicative of underlying immune maturation. These data support development of immune memory against SARS-CoV-2 providing insight into protection of the general unvaccinated part of the population.

The persistence of specific antibodies to SARS-CoV-2, the causative agent of COVID-19, is as of yet not fully understood. Partly because the follow-up time of studies investigating antibody kinetics is short owing to the novelty of the disease. Multiple studies show seroconversion to specific proteins following recent infection with SARS-CoV-2 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . Concurrently, studies report on the decay of antibodies over time, which raises the concern to what degree infected persons may remain protected to re-infection [4, 6, 8, 9, 11] . In addition, rapid decay of these antibodies would make seroprevalence estimates more difficult to interpret later after infection.

Specific antibodies are produced in different isotypes. Following most infections, immunoglobulin (Ig) M production is rapidly upregulated after infection and subsequently declines fast [13] [14] [15] . Specific IgA and IgG antibodies typically are initiated later than IgM production. In blood, IgG is the dominant circulating antibody isotype, whereas at mucosal surfaces, including the respiratory tract, IgA antibodies are more dominant [16] . The reported decay of SARS-CoV-2 antibodies will likely differ per isotype, necessitating detailed analyses of the distribution of different antibody isotypes over longer periods of time. The presence of antibodies longer after infection, and rapid upregulation of antibody secretion following re-infection depends on the presence of B cell memory. Memory B cells are responsible for the induction of high quality antibodies which are produced after class switching from IgM to IgG and require editing of the specificity of the antibody to provide an increased fit and binding strength of antibodies, collectively referred to as avidity maturation [17] . Hence, stronger avidity of antibodies is expected to be associated with an underlying cellular response, immune memory, and better ability to confer protection against future infection [18] . In addition to memory B cells, long-lived plasma cells contribute to the secretion of antibodies that can be detected multiple months and even years after an infection [19] .

Specifically, Spike S1-specific antibodies may neutralize the virus [1-3, 7, 20] , for which reason many vaccines aim to induce immunity to this part of the virus [21] . Understanding of anti-Spike antibody kinetics over prolonged periods of time is therefore of crucial importance [1, 5, 22, A c c e p t e d M a n u s c r i p t 6 23 ]. Very recent reports describe the presence of antibodies for up to or over 6 months post infection in specific populations such as health care workers or hospitalized patients [24, 25] . The duration of the antibody responses in the general population with generally mild symptoms however, has received little attention thus far.

Using samples of seroconverted individuals (N=353) from a nationwide prospective Pientercorona (PICO) serosurveillance study covering all ages, we studied the decay in SARS-CoV-2 Spike S1specific IgM, IgA and IgG antibodies over a period of 7 months after infection, and investigated the effect of COVID-19-related symptoms on antibody concentrations. In addition, we studied the development of avidity of anti-SARS-CoV-2 Spike S1 IgG antibodies as a marker of underlying cellular immunity and functionality of detected antibodies.

Participants from the PICO-serosurvey (design and inclusion are described in refs [7, 26, 27] ) were requested to return a self-collected finger-prick blood sample in a microtainer (SARSTEDT) by mail [7] . Participants were invited for a first round (PICO1) in April 2020 and for consecutive donations in June 2020 (PICO2) and October 2020 (PICO3). In the PICO2 round the study was extended with an additional nationwide random sample [28] . 365 participants seropositive for IgG to SARS-CoV-2 Spike S1 were available and because symptoms data were missing for 12 (3.3%) participants 353 were included in the present study. Since we aimed to study antibodies in the general population no other exclusion criteria were applied. Every study round, participants were asked to complete a questionnaire to collect type and date of onset of COVID-19-related symptoms 

Finger-prick blood samples were centrifuged and serum stored at -20 o C until analyses. The concentrations of IgG antibodies to SARS-CoV-2 Spike S1 (Wuhan isolate, GenBank #YP_009724390.1) were determined using a fluorescent bead-based immune assay as published previously [12] . which was further improved recently ( Figure S1 ). The assay selectively discriminates between antibodies to SARS-CoV-2 and the four known coronaviruses OC43, HKU-1, NL63 and 229E [12] . The specificity (99.7%) and sensitivity (91.6%) of the assay was determined using a heterogeneous sample including asymptomatic, and mild to severe COVID-19 cases as representative of COVID-19 cases in the general population. Since previous publication, the assay was extended to detect IgM and IgA antibodies to Spike S1 ( Figure S2 ). Thresholds for seropositivity were determined based on receiver operator curve analysis maximizing specificity and set at 1.20

Arbitrary Units/mL (AU/mL) for IgM, 0.50 AU/mL for IgA and 1.04 AU/mL for IgG.

Serum samples were diluted 1:200 and 1:8000 and incubated with Spike S1-coupled beads in SM01 buffer (Surmodics, USA) supplemented with 2% FCS while shaking (600 rpm) at room temperature for 45 mins. Next, plates were washed three times (PBS), incubated with PE-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories), IgA (Southern Biotech) or IgM (Jackson ImmunoResearch Laboratories) and incubated for an additional 30 mins. Samples were washed and acquired on a LX200 or FlexMap3D (Luminex). Concentrations were interpolated from an in-house reference consisting of pooled sera using a 5-parameter logistic fit. The coefficient of variation between independent assay runs ranges from 13.3-17.6.

Avidity of anti-Spike S1 IgG was performed on 73 samples of randomly-selected participants with varying concentrations of IgG by testing samples within the linear range of detection in the absence or presence of 1.1M of the chaotropic agent ammonium-thiocyanate [29, 30] . This concentration was confirmed to provide an optimal balance in discriminating antibodies of low and high avidity. Avidity is expressed as percentage of binding remaining when ammonium-thiocyanate is added.

A c c e p t e d M a n u s c r i p t 8

All statistical analyses were performed in R version 4.0.2 [31] . Participants with fever, dyspnoea, muscle ache, extreme tiredness, general malaise, painful respiration, joint pain, diarrhoea, and/or stomach ache were considered symptomatic for COVID-19. Asymptomatic participants and participants with mild -upper respiratory tract -complaints only (runny nose, sore throat, anosmia/ageusia, headache), were grouped together since these symptoms suggest contained, non-progressive infection. Sera of 365 participants were able, of which 12 were excluded because symptoms data were missing. Participants without symptom data were excluded (n=12). To study the change in the antibody concentrations and IgG avidity over time, antibody concentrations (AU/ml) were natural log-transformed and modelled separately. For each isotype, participants were included based on evidence of seroconversion to exclude persons that did not convert for IgM or IgA to influence decay rates (Table S1) . For IgG avidity all available data were used. Generalized estimating equations (GEE) with an exchangeable correlation structure was used to take into account correlation due to repeated sampling (using geepack version 1.3.1 [32] [33] [34] ). We selected the model with exponential decay over time if it resulted in a decrease in QIC (Quasilikelihood under Independence Model Criterion) of at least 2 compared to a model with a linear change over time [35] . Hereafter, age, sex, days since onset of symptoms, presence and duration of symptoms, and an interaction term between days since onset of symptoms and symptoms were included in the model as potential predictor variables. Age and duration of symptoms were A c c e p t e d M a n u s c r i p t 9 dichotomized at their median (i.e. 50+ years old vs. 49 or younger and 11 days or longer vs. 10 days or shorter, respectively). Variables with p<0.100 in univariable analyses were included in the multivariable model. Backwards selection was performed manually, excluding variables one-by-one with a p-value >0.050. Reported p-values are from model coefficients. The 2-fold decrease of IgG antibodies was calculated using the slope estimate and its 95% CI (i.e., -log(2)/slope) [29] .

Sera of 353 participants with specific IgG antibodies to spike S1 were available for analysis ( Figure 1A) . In total 738 samples of these participants were analysed, which are shown relative to date of onset of symptoms in Figure 1B . Table 1) . The median age was 48 (interquartile range 30-61) and 51 (IQR 32-66) years for symptomatic and asymptomatic/mildly symptomatic persons respectively. Of the symptomatic and asymptomatic/mildly symptomatic participants, 60% and 53% respectively were female. The most frequently reported symptoms were headache (67%), coughing (63%), fever (57%), muscle ache (52%) and general malaise (49%) while 35% reported dyspnea. Forty percent of those from the symptomatic participants group visited the general practitioner and 2% were admitted to the hospital.

The majority of individuals had anti-Spike S1 IgM (64%) and IgA (62%) antibodies in the first month after SARS-CoV-2 IgG seroconversion (Figure 2A) . The proportion of IgM-and IgA positive participants decreased after the first month to approximately 50% at 2-3 months post onset of symptoms. After 6 months since onset of symptoms 33% (95% CI 28-39) and 37% (95% CI 31-43) remained positive for IgM and IgA respectively. In the first month 99% of the participants were IgG A c c e p t e d M a n u s c r i p t 10 positive, which increased to 100% in months 2-3. After 6 months 92% (95% CI 89-95) were still positive for IgG.

Symptomatic individuals were more frequently positive for IgM or IgA in the first month after SARS-CoV-2 IgG seroconversion (Figure 2B and 2C, Table S2A ) compared with asymptomatic/mildly symptomatic persons. This difference gradually decreased over time, though it was still present after 6 months with 10% and 14% more symptomatic participants being positive for IgM and IgA respectively compared with asymptomatic/mildly symptomatic persons. IgG anti-Spike S1 seropositivity was observed regardless of COVID-19 symptoms. However, after 6 months, the individuals who had turned negative for IgG were mostly asymptomatic/mildly symptomatic: 87% positive for asymptomatic/mildly symptomatic persons versus 95% positive for symptomatic persons ( Figure 2D, Table S2A ).

Among persons who seroconverted to Spike S1 IgM (N=86), IgM concentrations showed a linear decline over time and were higher in symptomatic persons than asymptomatic/mildly symptomatic persons ( Figure 3A and Table S2B ). The average concentration of IgM decreased to the threshold for seropositivity after around 120 and 175 days for asymptomatic/mildly symptomatic, and more symptomatic individuals respectively. Among persons who seroconverted to Spike S1 IgA in the first 60 days after symptom onset (N=82), IgA concentrations showed an exponential decrease over time (Figure 3B) . The presence of symptoms resulted in higher IgA concentrations (Table S2B, S2C and S3). Average IgA concentration reached the threshold concentration after around 140 days.

IgG concentrations showed a linear decrease over time and symptomatic persons had significantly higher concentrations (Figure 3C and Table S2B ). The average concentrations of IgG did not intersect the threshold value for seropositivity within the studied timeframe of seven months post onset of A c c e p t e d M a n u s c r i p t 11 symptoms. IgM and IgA antibody concentrations over time for the entire study populationincluding those who did not seroconvert to IgM and IgA in the first 60 days following symptom onset -are shown in Table S2C . IgG and IgA, but not IgM, levels were higher in males and persons older than 50 years of age (Table S3 ). In addition, duration of symptoms for longer than 10 days resulted in increased IgG levels.

Since IgG antibodies persist we calculated the 2-fold decrease and measured avidity for IgG.

The 2-fold decrease of IgG concentrations, corrected for age, sex symptoms and duration of symptoms was estimated to be 158 days (95% CI 136-189 days). In addition to the duration of IgG in serum, we assessed the maturation of IgG to Spike S1 by assessing the avidity. The avidity index of Spike S1-specific IgG antibodies increased >2-fold during the seven months post onset of symptoms (p<0.015, Figure 3D) . Symptomatic individuals showed a stronger increase over time than asymptomatic/mildly symptomatic individuals (p=0.022, Table S3 ).

In light of the urgent question of the duration of immunity to SARS-CoV-2 following infection in the general population, we systematically studied the dynamics in seropositivity and concentrations of IgM, IgA and IgG antibodies to the SARS-CoV-2 Spike S1 protein among cases with different symptom profiles and investigated IgG maturation over time. Our data confirm that antibodies decline rapidly, in case of IgM and IgA isotypes. In contrast, 87% of the asymptomatic/mildly symptomatic and 95% of the symptomatic participants remained positive for A c c e p t e d M a n u s c r i p t 12 We showed that IgM and IgA antibodies start to decay within a few months post onset of symptoms, which may help explain the decline in seropositivity in some studies [6, 11, [13] [14] [15] . Since IgG antibodies persist much longer than IgM and IgA antibodies, the detection of IgG provides better sensitivity longer after infection, and therefore, IgG should be the isotype of choice in studies aiming to assess seroprevalence longer than 2 months after the infection and in longitudinal studies. IgG may also be the most informative for identifying memory induction, since specific IgG antibody development requires multiple cell divisions and class-switch recombination, processes that are a hallmark of memory formation. The hallmarks of memory formation -IgG antibodies with high avidity and persistence of antibodies -are presented in this study. The 2-fold decrease of IgG estimated in this study was 5-6-fold longer than the decay of passively transferred maternal antibodies [29, 37, 38] . This decrease rate may still be underestimated since the decay of antibodies is the most pronounced in the first months after the induction of the antibodies. Therefore, longer follow-up studies should re-assess the persistence of antibodies to Spike S1 of SARS-CoV-2 and compare these to persistence as observed for other viruses [39, 40] .

The da formation of B cell memory, implies that antibodies can be rapidly upregulated in response to re-infection in order to effectively control the virus [18, 41] . It is still unknown which antibody levels confer protection against re-infection or COVID-19 disease. While the antibodies detected in this study are restricted to Spike S1, we cannot exclude the detection of antibodies not necessarily contributing to virus neutralization. In light of newly emerging strains with mutations that may escape neutralization by antibodies the cross-protection by pre-existing immunity either through infection or vaccination need to be closely monitored. Interestingly, having had COVID-19like symptoms resulted in higher antibody concentrations for IgG and IgM and faster development of IgG avidity, compared with persons who remained asymptomatic/mildly symptomatic after SARS-CoV-2 infection. The reason for this may be a stronger inflammatory response, a higher or longer viral replication period, or both, that may result in better and longer-lasting immunity.

A c c e p t e d M a n u s c r i p t 13 This study is unique in analyzing samples collected in the general population including all ages and COVID-19 disease severities. While the findings reflect SARS-CoV-2 antibody dynamics of the general public, the study has several limitations. Participants were included based on IgG anti-Spike S1 seropositivity, and therefore we may have missed a few persons that seroconverted for IgM or IgA, but not, or insufficiently, for IgG. The time since onset of COVID-19 was based on selfreported symptoms on a presumed SARS-CoV-2 infection, and therefore may be less accurate since symptoms could be caused by other infections still prevailing during the peak of the epidemic.

However, the reported date of onset of symptoms of the participants matched the national epidemiological data of COVID-19 cases in the Netherlands [36] . In addition, the paired samples of seroconverted individuals collected 6 months apart confirm that IgG antibodies persist for more than 6 months in 92% of seroconverted individuals [42] . Despite the persistence of IgG antibodies, the decay cannot be neglected and will eventually result in an underestimation of the proportion of infected persons in the population once this proportion has crossed the cut-off levels of specific antibody detection.

In conclusion, our analyses included 353 individuals participating in a nationwide population study with 7 months follow-up for most participants, which is a substantially longer follow-up period than most other population studies [3, 10] . We show that anti-SARS-CoV-2 Spike S1 IgG antibodies persist for extended time, i.e. longer than six months. Therefore, we propose that analysis of IgG anti-Spike S1 of SARS-CoV-2 will generate the most consistent seroprevalence estimates and provide understanding of the duration of protective immunity. In view of an IgG decay-rate 5-6 -fold slower than reported for passively transferred maternal IgG and the improving IgG avidity over time, B-cell memory is likely established in most individuals. In addition, our data suggest that the duration of the IgG response is likely longer for symptomatic COVID-19 cases due to higher initial concentrations. Our results aid the interpretation of the duration of immunity in unvaccinated persons and provide a framework for the evaluation of immunity induced by vaccines for SARS-CoV-

A c c e p t e d M a n u s c r i p t 14 NOTES M a n u s c r i p t 18 

Duration of serum neutralizing antibodies for SARS-CoV-2: Lessons from SARS-CoV infection

Dynamics of neutralizing antibody titers in the months after SARS-CoV-2 infection

Humoral Immune Response to SARS-CoV-2 in Iceland

Longitudinal Change of Severe Acute Respiratory Syndrome Coronavirus 2 Antibodies in Patients with Coronavirus Disease

Longitudinal Dynamics of the Neutralizing Antibody Response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection

Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans

Nationwide seroprevalence of SARS-CoV-2 and identification of risk factors in the general population of the Netherlands during the first epidemic wave

Neutralizing Antibody Responses to Severe Acute Respiratory Syndrome Coronavirus 2 in Coronavirus Disease 2019 Inpatients and Convalescent Patients

Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients

Antibodies in Persons with Mild Covid-19

SARS-CoV-2-specific antibody detection for seroepidemiology: a multiplex analysis approach accounting for accurate seroprevalence

The antibody response to primary and secondary infection with respiratory syncytial virus: Kinetics of class-specific responses

Chronological evolution of IgM, IgA, IgG and neutralisation antibodies after infection with SARS-associated coronavirus

Kinetics of influenza A virus nucleoprotein antibody (IgM, IgA, and IgG) in serum and oral fluid specimens from pigs infected under experimental conditions

The immune geography of IgA induction and function

Mechanism and Regulation of Class Switch Recombination

Generation of memory B cells and their reactivation

Plasma cell survival in the absence of B cell memory

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model

Immunesurveillance for vaccine-preventable diseases

Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses

The duration, dynamics and determinants of SARS-CoV-2 antibody responses in individual healthcare workers

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection

Third national biobank for population-based seroprevalence studies in the Netherlands, including the Caribbean Netherlands

Associations between measures of social distancing and SARS-CoV-2 seropositivity: a nationwide population-based study in the Netherlands

Antibody Responses to Respiratory Syncytial Virus: A Cross-Sectional Serosurveillance Study in the Dutch Population Focusing on Infants Younger Than 2 Years

Characteristics of HPV-Specific Antibody Responses Induced by Infection and Vaccination: Cross-Reactivity, Neutralizing Activity, Avidity and IgG Subclasses

R: A language and environment for statistical computing

The R package geepack for generalized estimating equations

Estimating equations for association structures

Geepack: yet another package for generalized estimating equations

Model Selection and Multimodel Inference A Practical Information-Theoretic Approach

Half-life of the maternal IgG1 allotype in infants

Waning of maternal antibodies against measles, mumps, rubella, and varicella in communities with contrasting vaccination coverage

Duration of Humoral Immunity to Common Viral and Vaccine Antigens

Heterogeneity and longevity of antibody memory to viruses and vaccines

High Concentrations of Measles Neutralizing Antibodies and High-Avidity Measles IgG Accurately Identify Measles Reinfection Cases. Clinical and vaccine immunology

Orthogonal SARS-CoV-2 Serological Assays Enable Surveillance of Low-Prevalence Communities and Reveal Durable Humoral Immunity

Male % (n) 40% (85) 47% (65)

The authors acknowledge the study participants. We also acknowledge Patricia Kaaijk, Marjan Bogaard, Marjan Kuijer, Debbie van Rooijen, Pieter van Gageldonk and Irina Tcherniaeva for assisting in the execution of the study. The authors acknowledge Willem Luijtjes for critically reviewing the manuscript.

This work was supported by the National Institute for Public Health and the Environment, The Netherlands (no grant number available).

A c c e p t e d M a n u s c r i p t 15 A c c e p t e d M a n u s c r i p t 19 Median duration of symptoms*, IQR 11, 6-18 6, 2-9 *Data on the duration of symptoms was available for 153 participants in the symptomatic group and 26 participants in the asymptomatic/only mild upper respiratory symptoms group. # participants with these symptoms are included in the 'symptomatic' group and therefore 'NA' in the 'asymptomatic / only mild respiratory symptoms' group. Table S3 ).