key: cord-0770630-lchooiqq
authors: Lumley, Sheila F; Wei, Jia; O’Donnell, Denise; Stoesser, Nicole E; Matthews, Philippa C; Howarth, Alison; Hatch, Stephanie B; Marsden, Brian D; Cox, Stuart; James, Tim; Peck, Liam J; Ritter, Thomas G; de Toledo, Zoe; Cornall, Richard J; Jones, E Yvonne; Stuart, David I; Screaton, Gavin; Ebner, Daniel; Hoosdally, Sarah; Crook, Derrick W; Conlon, Christopher P; Pouwels, Koen B; Walker, A Sarah; Peto, Tim E A; Walker, Timothy M; Jeffery, Katie; Eyre, David W
title: The duration, dynamics and determinants of SARS-CoV-2 antibody responses in individual healthcare workers
date: 2021-01-06
journal: Clin Infect Dis
DOI: 10.1093/cid/ciab004
sha: f08dbb992c4a77e882201f15d892228789bca6a7
doc_id: 770630
cord_uid: lchooiqq

BACKGROUND: SARS-CoV-2 IgG antibody measurements can be used to estimate the proportion of a population exposed or infected and may be informative about the risk of future infection. Previous estimates of the duration of antibody responses vary. METHODS: We present 6 months of data from a longitudinal seroprevalence study of 3276 UK healthcare workers (HCWs). Serial measurements of SARS-CoV-2 anti-nucleocapsid and anti-spike IgG were obtained. Interval censored survival analysis was used to investigate the duration of detectable responses. Additionally, Bayesian mixed linear models were used to investigate anti-nucleocapsid waning. RESULTS: Anti-spike IgG levels remained stably detected after a positive result, e.g., in 94% (95% credibility interval, CrI, 91-96%) of HCWs at 180 days. Anti-nucleocapsid IgG levels rose to a peak at 24 (95% credibility interval, CrI 19-31) days post first PCR-positive test, before beginning to fall. Considering 452 anti-nucleocapsid seropositive HCWs over a median of 121 days from their maximum positive IgG titre, the mean estimated antibody half-life was 85 (95%CrI, 81-90) days. Higher maximum observed anti-nucleocapsid titres were associated with longer estimated antibody half-lives. Increasing age, Asian ethnicity and prior self-reported symptoms were independently associated with higher maximum anti-nucleocapsid levels and increasing age and a positive PCR test undertaken for symptoms with longer anti-nucleocapsid half-lives. CONCLUSION: SARS-CoV-2 anti-nucleocapsid antibodies wane within months, and faster in younger adults and those without symptoms. However, anti-spike IgG remains stably detected. Ongoing longitudinal studies are required to track the long-term duration of antibody levels and their association with immunity to SARS-CoV-2 reinfection.

Measurable IgG antibodies to SARS-CoV-2 antigens develop after many, but not all, SARS-CoV-2 infections. [1] [2] [3] [4] Serological responses are typically detectable within 1-3 weeks. [5] [6] [7] [8] This allows antibody assays to be used to estimate the proportion of a population exposed or infected.

Additionally, although the extent of immunity associated with different antibody titres and other immune responses is yet to be fully determined, it is probable that antibody levels will provide some information about the risk and/or severity of future infection.

However, SARS-CoV-2 IgG antibody levels are dynamic over time. [9] This has implications for epidemiological studies, e.g., if IgG levels fall below detection thresholds before they are measured, past infections may be under ascertained. Similarly, it has implications for estimating population protection if antibodies are a marker for protective immunity.

Contrasting data have been made available on the longitudinal trajectory and longevity of antibodies induced by SARS-CoV-2 infection. For example, a US study showed IgG antibody levels to trimerised spike were relatively stable in 121 individuals around 110 days post symptom onset. [10] Similarly, data from 1215 individuals in Iceland suggest IgG responses to nucleocapsid and the S1 component of spike were sustained for 100-125 days. [11] However, others have noted declines in neutralizing antibodies over similar time periods. [12] [13] [14] We have recently undertaken baseline serological testing in a cohort of >10,000 HCWs. [15] We now describe serial SARS-CoV-2 antibody measurements, demonstrating quantitative anti-nucleocapsid responses fall over time and vary with age, ethnicity and previous symptoms, but anti-spike levels antibodies remain stably detected in most individuals.

Oxford University Hospitals (OUH) offers both symptomatic and asymptomatic SARS-CoV-2 testing programmes to staff at its four teaching hospitals in Oxfordshire, UK. 12,411 healthcare workers (HCWs) have undergone serological testing to date; data on HCWs who attended more than once for antibody testing are presented.

SARS-CoV-2 PCR testing of nasal and oropharyngeal swabs for all symptomatic (new persistent cough, fever ≥37.8C, anosmia/ageusia) staff was offered from 27-March-2020 onwards.

Asymptomatic HCWs were invited to participate in voluntary staff testing for SARS-CoV-2 by nasal A c c e p t e d M a n u s c r i p t 5 and oropharyngeal swab PCR and serological testing from 23-April-2020 onwards. The cohort, associated methods and findings from the first test per individual have been previously described. [15] Following initial PCR and antibody testing, asymptomatic HCWs were invited to optionally attend for serological testing up to once every two months, with some offered more frequent screening as part of related studies. Asymptomatic staff were also offered optional SARS-CoV-2 PCR tests every two weeks.

Serology for SARS-CoV-2 IgG to nucleocapsid protein was performed using the Abbott Architect i2000 chemiluminescent microparticle immunoassay (CMIA; Abbott, Maidenhead, UK). Antibody levels ≥1.40 manufacturer's arbitrary units were considered positive, 0.50-1.39 equivocal (following Abbott Diagnostics Product Information Letter PI1060-2020) and <0.5 negative. Anti-trimeric-spike IgG levels were measured using an ELISA developed by the University of Oxford, [2] using netnormalised signal cut-off of ≥8 million units to determine antibody presence and defining 4.0-7.9 million units as equivocal. [16] Details on PCR assays are provided in the Supplement.

For anti-nucleocapsid antibodies, individuals with ≥1 positive antibody result (titre ≥1.40) and ≥2 antibody results were classified as showing rising titres only, falling or stable titres only, or both.

Those with only one measurement could not be classified and were excluded. In those with falling/stable titres we estimated the duration of antibody responses following the maximum observed result using Bayesian linear mixed models and their association with age, gender, ethnicity, previous self-reported symptoms and PCR results (allowing correlated random intercept and slope terms, Supplement, Table S1 ). We assumed antibody levels fell exponentially, and so modelled log2 transformed antibody levels over time (observed data and fitted models demonstrated close congruence, Supplementary File). The incidence of Covid-19 in our hospital fell after a peak in March and April 2020, [15] such that re-exposure of HCWs was uncommon; we therefore had insufficient data to study boosting of antibody responses.

We additionally modelled the antibody trajectory from a first positive PCR test using a similar approach but allowing for non-linear effects of time rather than assuming an exponential decline.

It was not possible to model anti-spike IgG titres over time in those with a positive result as most positive readings were above the upper limit of quantification of the assay (9 million units).

Therefore, we considered changes in binary results for both anti-spike and anti-nucleocapsid using Bayesian interval censored regression to estimate the proportion of individuals remaining antibody 

Among 522 individuals with ≥1 anti-nucleocapsid IgG-positive sample, 70 (13%) seroconverted with rising titres only and so were excluded from analyses of the duration of response following a peak IgG result (39/70 had a PCR test and are included in a separate analysis below). In the remaining 452 (87%), the median (IQR) [range] number of samples tested was 2 (2-3) [2] [3] [4] [5] and time from the first to last sample was 121 (83-143) days. Only 3/120 (3%) individuals with ≥3 measurements had a final titre above the minimum observed, i.e. potential evidence of boosting, and titre increases were all <5%. The median (IQR) age was 41 (29-50) years and 75% of participants were female ( Table   1) . The most common self-reported ethnic groups were White (302, 67%) and Asian (89, 20%;

predominately south Asian and Filipino). 274 (61%) recalled self-identified Covid-19-like symptoms between 01-February-2020 and testing. 95 (21%) had a positive SARS-CoV-2 PCR following symptomatic testing and 59 (13%) a positive PCR during asymptomatic screening. It is likely that A c c e p t e d M a n u s c r i p t 7 many of the remainder were infected prior to widespread availability of testing. The first positive PCR in each individual was prior to or on the day of their maximum antibody titre in all but 5/154 (3%, tested 3-17 days later).

Using a Bayesian statistical model, the trajectory of anti-nucleocapsid IgG levels following the maximum measured titre in each individual is shown in Figure 3A . The estimated mean antibody half-life was 85 (95% credibility interval, CrI 81-90) days and estimated mean maximum antibody 

Within this cohort of HCWs of working age, age, self-reported ethnicity, prior symptoms compatible with Covid-19 and a positive SARS-CoV-2 PCR were independently associated with changes in antinucleocapsid trajectories ( (98%) had ≥1 antibody test ≥14 days after their PCR-positive test, and in 29 (56%) this was before 90 days. PCR cycle threshold values were lower in individuals who seroconverted (Table S4) .

Data from PCR-positive individuals who seroconverted were used to model antibody trajectories relative to a first positive PCR test. Antibody levels rose to a peak at 24 (95%CrI 19-31) days post-first positive PCR test, before beginning to fall ( Figure S6 ). Comparing with the antibody trajectory estimated in the main analysis, the estimated rates of waning were consistent between the two models. Antibody trajectories were similar in those being tested following symptoms or during asymptomatic screening (Figure 4 ). IgG waning and reinfection within a year is reported for seasonal coronaviruses [17] whereas IgG remains detectable against SARS-CoV and MERS-CoV 1-3 years later. [18] The differences we observe in SARS-CoV-2 antibody trajectories may be antigen and/or assay dependent, e.g., stable anti-spike antibodies with waning of anti-nucleocapsid IgG using the same Abbott platform as in our study was also seen in an earlier smaller study, but total anti-nucleocapsid antibodies assayed using a Roche platform remained stable. [14] To some extent these findings are conditional on assay cut-offs which can be tuned to prioritise sensitivity or specificity, with inherently more specific assays having potential to also be set more sensitively, resulting in apparently longer durations of detectable antibody responses.

For anti-nucleocapsid, we observe higher IgG titres with longer durability occurring after symptomatic PCR-positive infection, consistent with data from Long et al. where 40% of asymptomatic individuals and 13% of the symptomatic group became negative for IgG in the early convalescent phase [19] and consistent with emerging coronaviruses, where antibody titres remained detectable longer after more severe illness, [18] waning more rapidly after asymptomatic infection.

Relatively short-term anti-nucleocapsid IgG responses have two epidemiological consequences.

Firstly, antibody waning may lead to under-ascertainment of previous infections within the current pandemic, particularly in younger individuals following asymptomatic/mild infection. Additionally, IgG testing is unlikely to determine whether SARS-CoV-2 has circulated historically, e.g. in a particular geographic region.

Older age (within this cohort of working age HCWs, up to 69 years) was associated with higher maximum observed anti-nucleocapsid IgG titres and longer half-lives, with similar findings associated with Asian ethnicity (many of the Asian HCWs in our study came to work in the UK healthcare system as adults). It is possible to hypothesise that this could arise from boosting of cross-reactive antinucleocapsid antibodies from prior exposure, e.g. to a previously circulating or geographicallyrestricted human coronavirus. However, anti-nucleocapsid cross-reactivity between endemic coronaviruses and other epidemic coronaviruses, SARS-CoV and MERS-CoV, is uncommon. [18, 21] A c c e p t e d M a n u s c r i p t 10 

The assays we used could only measure quantitative trajectories for anti-nucleocapsid IgG, further studies, e.g. using multiple serum dilutions, are required to quantify anti-spike IgG over time. We therefore cannot say whether the longer duration of positive anti-spike responses is due to slower waning or higher initial levels relative to assay cut-offs. Another limitation of our study was that our cohort of individuals consisted of adults of working age (17-69 years); further longitudinal studies will be required to investigate younger and older age groups. The small numbers of self-reported Black (n=25) and Other (n=36) ethnicities reduced/limited power to detect an association between these ethnicities and antibody trajectories. We also do not account for mediators, e.g. socioeconomic inequalities, that may link ethnicity to antibody responses in the absence of a direct causal relationship. Due to many of our staff developing symptoms before widespread SARS-CoV-2 PCR testing was available, only 34% of the anti-nucleocapsid-positive cohort had a documented positive PCR, and as a proportion of the cohort were asymptomatic throughout, we modelled time from maximum positive antibody test rather than time from first positive PCR or time from symptom onset in our main analysis of antibody durability. However, under an exponential assumption, halflives can be unbiasedly estimated from any measurements taken after a maximum; we excluded individuals with only evidence of rising titres to avoid underestimating half-lives. Further, data from those that were PCR positive were consistent with this analysis (Figure 4) .

Multiple different assays are in use globally to characterize antibody responses to SARS-CoV-2. Here we study only two; however, other antibody classes and targets, and aspects of immunity, including the innate and cellular responses are important in conferring post-infection immunity. [22] When comparing longitudinal studies of antibody durability, care must be taken, as the various assays have not yet been cross-calibrated, and implications for protective immunity are not fully understood.

It is widely recognized that pathogen-specific IgG levels decline after the acute phase of an infection.

After the initial humoral response in which short-lived plasmablasts secrete high titres of antibody, long-lived plasma cells and memory B cells then contribute to longer-term antibody-mediated protection. [23] Although declines in IgG titres are expected, understanding the assay-dependent rate of decline, whether and when titres fall below assay positive cut-offs, and how these titres relate to protection from subsequent asymptomatic and symptomatic re-infection is crucial.

A c c e p t e d M a n u s c r i p t 11 Serological testing also helps quantify the extent of infection in populations, informing epidemiological models and public health strategies. However waning antibody levels may lead to underestimated exposure due to loss of seropositivity. For example, using the anti-nucleocapsid assay an estimated 33% of individuals seroreverted (i.e. fell below the positive cut off for the Abbott assay) within 3 months of IgG detection and an estimated 53% by 6 months ( Figure 5B ). Therefore, depending on the assay used, sero-epidemiological surveys performed several months into this pandemic may have underestimated prior exposure, especially in younger adults who lose detectable antibody faster. Our findings contrast with the repeated cross-sectional REACT2 study, [24] which reported greater reductions over time at a population-level in the proportion of adults ≥65 years testing antibody-positive, and more sustained responses in those 18-24 years.

Nearly all HCWs in our study were <65 years, other differences may arise from study design, the assay used, and the potential for new infections predominantly in younger people [25] to replace others who had sero-reverted, supporting population-level seroprevalence (in our study each individual was followed-up separately). 85 Table 2 . Univariable and multivariable models of determinants of SARS-CoV-2 anti-nucleocapsid antibody trajectories. Posterior mean and 95% credibility intervals for the maximum antibody level

Antibody tests for identification of current and past infection with SARS-CoV-2

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SARS-CoV-2-IgG response is different in COVID-19 outpatients and asymptomatic contact persons

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Stringent thresholds for SARS-CoV-2 IgG assays result in under-detection of cases reporting loss of taste/smell

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We thank all OUH staff who participated in the staff testing programme, and the staff and medical students who ran the programme. This work uses data provided by healthcare workers and collected by the UK's National Health Service as part of their care and support. We thank all the people of