key: cord-351278-nm2bq717
authors: Thompson, Craig; Grayson, Nicholas; Paton, Robert; Lourenço, José; Penman, Bridget; Lee, Lian Ni; Odon, Valerie; Mongkolsapaya, Juthathip; Chinnakannan, Senthil; Dejnirattisai, Wanwisa; Edmans, Matthew; Fyfe, Alexander; Imlach, Carol; Kooblall, Kreepa; Lim, Nicholas; Liu, Chang; Lopez-Camacho, Cesar; McInally, Carol-Anne; Ramamurthy, Narayan; Ratcliff, Jeremy; Supasa, Piyada; Wang, Beibei; Mentzer, Alexander J; Turner, Marc; Semple, Calum; Baillie, John Kenneth; Harvala, Heli; Screaton, Gavin; Temperton, Nigel; Klenerman, Paul; Jarvis, Lisa; Gupta, Sunetra; Simmonds, Peter
title: Neutralising antibodies to SARS coronavirus 2 in Scottish blood donors - a pilot study of the value of serology to determine population exposure
date: 2020-04-17
journal: nan
DOI: 10.1101/2020.04.13.20060467
sha: 
doc_id: 351278
cord_uid: nm2bq717

Background. The extent of spread of SARS coronavirus 2 (SARS-CoV-2) in the UK and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of recent blood donors in Scotland to detect antibodies to SARS-CoV-2 as a marker of past infection. Methods. A pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study group comprised samples from 1000 blood donors collected in Scotland during March, 2020. Controls were collected from 100 donors in Scotland during 2019. Findings. All samples collected on the 17th March, 2020 (n=500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in 5 of the 500 samples collected 21st to 23rd March; one further sample was reactive in an anti-spike ELISA. Interpretation. Although we cannot use the rise in numbers seropositive to infer the contemporary seroprevalence or the growth rate of the epidemic, we note that they are consistent with frequency of reported diagnosed infections and SARS-CoV-2-associated deaths reported in that time period in Scotland, given that seroconversion takes up to 2-3 weeks. It should also be noted that blood donors are not representative of the general population; in particular, those with a history of recent respiratory infections are deferred. Finally, it is unknown what proportion of infected individuals seroconvert and become reactive in the assays used. Serial follow up studies are needed to track infection and seroconversion in this and other similar populations However, these data indicate that sero-surveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic like the current SARS-CoV-2 outbreak.

SARS-CoV-2 as a marker of past infection. Interpretation. Although we cannot use the rise in numbers seropositive to infer the contemporary seroprevalence or the growth rate of the epidemic, we note that they are consistent with frequency of reported diagnosed infections and SARS-CoV-2-associated 60 deaths reported in that time period in Scotland, given that seroconversion takes up to [2] [3] weeks. It should also be noted that blood donors are not representative of the general population; in particular, those with a history of recent respiratory infections are deferred. [1] [2] [3] [4] . Since the first reports in December, 2019, infections with SARS-CoV-2 have been reported from an increasing number of countries worldwide, with particularly high incidence of diagnosed infections and associated deaths from respiratory disease initially in China but more recently in Italy, Iran, Spain, France and the USA (https://www.who.int/emergencies/diseases/novel-coronavirus-2019). Increasing age, male 80 gender, smoking and comorbidities such as cardiac disease, hypertension and diabetes have been identified as risk factors for severe infections [5] [6] [7] . For as yet unknown reasons, infants and children seem to be less at risk for moderate to severe COVID-19 disease 8 .

Compared to Italy and Spain, the SARS-CoV-2 pandemic was at a relatively early stage in 85 the UK in early March 2020. However, a dramatic rise in the number of admissions of patients with severe SARS-CoV-2 infections followed and emergency plans have been implemented. Prediction of the future severity of the outbreak, and most specifically the trajectory of severe cases that require hospitalisation and intensive care support is key to the national response, as it is throughout several affected countries worldwide. Predicting disease 90 outcomes is complex; in addition to the basic information of the proportions of individuals in different age ranges who develop severe disease, the severity of the outbreak is also crucially dependent on current population immunity and virus transmissibility. Mean estimates of R0 of 3.29 (2.12 -4.45 interquartile range from 12 studies based on presumed SARS-CoV-2 naive populations) 9 indicate that a 50%-75% minimum level of herd immunity from past 95 infection is sufficient for a sustained reduction of new infections with time. SARS-CoV-2 spread may be further influenced by seasonal changes in transmissibility, as observed for other respiratory coronaviruses infecting humans [10] [11] [12] .

In the current study we have taken the first steps towards estimating SARS-CoV-2 exposure 100 in a European country by measuring seroprevalence in a sample of blood donations. Samples from donors in an age range from 18-75 years collected across Scotland on the 17 th March and 21 st -23 rd of March, 2020 were assayed for neutralising antibody to SARS-CoV-2 using a pseudotyped SARS-CoV-2 virus microneutralisation (pMN) assay format used previously for SARS-CoV-1 and Ebolavirus seroepidemiology purposes [13] [14] [15] and confirmed using an 105 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04. 13.20060467 doi: medRxiv preprint During the assay, plates were barcoded and controls were spaced throughout the runs.

Individuals were blinded regarding the arrangement of spaced positive controls on the plates.

Enzyme-linked immunosorbent assay (ELISA). Antibodies to the trimeric S protein were detected by ELISA. MAXISORP immunoplates (442404; NUNC) were coated with 145

StrepMAB-Classic (2-1507-001;iba). Plates were blocked with 2% skimmed milk in PBS for one hour and then incubated with 0.125ug of soluble trimeric SARS-CoV2 trimeric Spike protein or 2% skimmed milk in PBS. After one hour, plasma was added at 1:50 dilution, followed by ALP-conjugated anti-human IgG (A9544; Sigma) at 1:10,000 dilution or ALPconjugated anti-human IgM (A9794; Sigma) at 1:5,000 dilution. The reaction was developed 150 by the addition of PNPP substrate and stopped with NaOH. The absorbance was measured at 405nm after 1 hour.

Statistical and Modelling Analysis. Samples were analysed following the protocol outlined in Ferrara & Temperton 16 which was implemented using the SciPy curve fit 15 . Values for 155 each plate ( ) were corrected for background signal by subtracting the average of 6 negative control wells ( ). The standardised percentage neutralisation for each sample value ( ) was calculated by benchmarking against the average of 6 technical positive controls for each plate ( ̅ ) using the equation:

Standardised percentages from two replicates of the same sample were averaged for each dilution. A three-parameter logistic function was then fit to these averages using non-linear least squares: 165

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The copyright holder for this preprint . https://doi.org/10.1101/2020.04. 13.20060467 doi: medRxiv preprint Here, the averaged standardised neutralisation percentage ̅ ), is a function of the logarithm 170 (base 10) of the dilution factor and the 50% inhibitory concentration . The value corresponds to the dilution factor where 50% neutralisation is predicted to be achieved.

Larger values correspond to samples with stronger antibody responses which require higher dilutions to reduce neutralisation. The parameter describes the asymptote of the curve and was fixed to 100 (100% neutralisation was the maximum). The parameter " 175 describes the steepness of the slope for the sigmoidal curve and was restricted to take values < 0 (only negative responses of neutralisation to dose were permitted). The quality of the (Inverness Health Board) areas. There was an approximately equal representation of males and females amongst donors but a restricted age range from 18 (minimum donor age) to 75 ( Fig. 1) ; this was also skewed towards older age ranges compared to the Scottish population within that range ( Fig. S1 ; Suppl. Data).

Samples from both weeks in 2020 and the 2019 controls were assayed by pMN assays using a two-fold dilution series of plasma from 1:20 -1:640. The reduction in luciferase signal . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.04. 13.20060467 doi: medRxiv preprint through antibody binding at each dilution was curve-fitted in order to estimate the dilution leading to a 50% inhibition of pseudotype replication ( ). A second metric is the degree of fit of the datapoints from the dilution series to the predicted sigmoid curve using for fitting, 205 expressed as R 2 . Samples showing non-specific blocking of entry (ie. non-concentration dependent) will show a low R 2 value. Of the 7 positive control samples, 6 possessed detectable neutralising ability, above the range seen in the pre-pandemic samples, and good is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.04. 13.20060467 doi: medRxiv preprint in Scotland at the time of sample collection. In interpreting the significance of this seroprevalence, we review several factors which may influence antibody detection rates and how this might translate into calculations of population exposure and immunity and projections of the outcomes of the SARS-CoV-2 outbreak. 240

The representativeness of the study region. The study was based upon testing anonymised samples from blood donors collected in March, 2020. Scotland has a population of 5.4 million and a relatively low population density (67.2/km 2 ). However, it contains several large cities (Glasgow, Edinburgh) which geospatially would support transmission networks typical 245 of much of Western Europe. Public health data for COVID-19 cases indicated that by the 26 th March, shortly after donor collection, a total of 894 infections had been diagnosed and 25 deaths recorded (normalised incidence: 0.46/100,000). This compares to an overall confirmed incidence for the UK of 0.62/100,000 on the same day. The rate in Scotland was higher than Germany (0.18/100,000), comparable to Denmark (0.41/100,000), France (0.30/100,000) and 250

Spain (0.42/100,000) but substantially lower than Italy (11.1/100,000). The value of these comparisons has to be tempered by potential differences between countries in criteria used to attribute death from SARS-CoV-2 infection. Furthermore the situation is highly dynamicon the 1 st April, the rate in Scotland had risen to 0.87/100,000 but it was much higher in England (2.5/100,000) and rates may diverge further in the future. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.04.13.20060467 doi: medRxiv preprint comprehensive analysis published by China CDC 17 estimates that 81% of those infected will have mild or inapparent disease (symptoms of upper respiratory tract viral infection, mild fever, cough (dry), sore throat, nasal congestion, malaise, headache, muscle pain but without dyspnoea or other sign of respiratory distress or insufficiency). Given that these symptoms do not differ substantially from other winter respiratory infections, it is likely that a substantial 275 proportion of the estimated 81% of mild cases would proceed to donate.

Sensitivity and specificity of the serological tests used. There are currently no agreed standards or controls available for serological testing for SARS-CoV-2. The results presented in the study are therefore based on a formally non-validated assay. However, we believe that 280 the neutralisation antibody test is likely to be robust. Firstly, it is based on the same design as pMN assays for SARS-CoV-1, ebolavirus and influenza A virus; pMN assays that have demonstrated high specificity for target virus neutralising antibodies and a sensitivity that is often greater than achieved in neutralising antibody assays for whole virus [13] [14] [15] . Overall, although sampling was limited, there was concordance between the pMN assay to detect 285 neutralising antibody and the detection of anti-spike IgG antibody by ELISA . The pMN assay does, however, require comparison with neutralisation of live virus for further interpretation of these data.

The specificity of both assays is potentially influenced by cross-reactivity with other human 290 coronaviruses, including the respiratory viruses, OC43 and HKUI in the Betacoronavirus genus that circulate in winter months. Some previous studies have indicated an absence of reactivity of negative control sera against the SARS-CoV-1 spike protein in ELISA and the high specificity of assays based on the spike protein compared to those using the more conserved nucleoprotein or whole virus 18 . SARS-CoV-2 spike protein is likely to share this 295 specificity. Existing data on neutralising antibodies indicates that assay specificity may be even greater; although both SARS-CoV-1 and -2 enter cells through the ACE-2 receptor [19] [20] [21] , and possess structurally similar spike proteins 20,22-24 , it has recently been demonstrated that neutralising monoclonal antibodies (MAbs) against SARS-CoV-1 infection bound but did not neutralise SARS-CoV-2 25 , Conversely, MAbs raised against SAR-CoV-2 showed little or no 300 cross-neutralisation of SARS-CoV-1 26,27 . However, cross-reactivity is most unlikely to cause an assay specificity problem, as SARS-CoV-1 has never spread significantly in the UK or elsewhere in Europe 28 .

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The copyright holder for this preprint . https://doi.org/10.1101/2020.04. 13.20060467 doi: medRxiv preprint

Serological tests used for determining population exposure are based upon an assumption of a durable virus-specific IgG antibody response to infection that persists for years after infection. This is typically assumed in seroprevalence studies for many human pathogens, including poliovirus, measles, and hepatitis B virus. There is little information, however, on is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint . https://doi.org/10.1101/2020.04.13.20060467 doi: medRxiv preprint generate antibody detectable in a commercial ELISA test 39 . Technical considerations aside, the temporal gap between exposure and seroconversion, coupled with the rapid spread of this virus means that during the initial phase of the epidemic an important difference will exist -at 340 any given time point -between the fraction of those recently exposed/infected and the fraction of those who have seroconverted . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.13.20060467 doi: medRxiv preprint The numbers of seropositive blood donor samples in the study divided regionally by health board. Sampling density is indicated by the shading of each region. The locations of the seropositive donors are indicated in red text, arrows and borders. Histograms show the sampling frequencies of different age classes for week 1 (blue) and week 2 (grey) for each health board. 505 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04.13.20060467 doi: medRxiv preprint Post-pandemic positive ELISA tested samples are indicated by by filled red squares.. The dotted line indicates the highest IC50 value found among the pre-pandemic samples.

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(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04. 13.20060467 doi: medRxiv preprint 

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.04. 13.20060467 doi: medRxiv preprint 

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