key: cord-0808873-bgdz6mf3
authors: Harvala, Heli; Robb, Matthew L.; Watkins, Nick; Ijaz, Samreen; Dicks, Steven; Patel, Monika; Supasa, Piyada; Wanwisa, Dejnirattisai; Liu, Chang; Mongkolsapaya, Juthathip; Bown, Abbie; Bailey, Daniel; Vipond, Richard; Grayson, Nicholas; Temperton, Nigel; Gupta, Sunetra; Ploeg, Rutger J.; Bolton, Jai; Fyfe, Alex; Gopal, Robin; Simmonds, Peter; Screaton, Gavin; Thompson, Craig; Brooks, Tim; Zambon, Maria; Miflin, Gail; Roberts, David J.
title: Convalescent plasma therapy for the treatment of patients with COVID‐19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels
date: 2020-12-17
journal: Transfus Med
DOI: 10.1111/tme.12746
sha: 1fc4663289c983640ea847f7685c5391fac18832
doc_id: 808873
cord_uid: bgdz6mf3

INTRODUCTION: The lack of approved specific therapeutic agents to treat coronavirus disease (COVID‐19) associated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection has led to the rapid implementation of convalescent plasma therapy (CPT) trials in many countries, including the United Kingdom. Effective CPT is likely to require high titres of neutralising antibody (nAb) in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS‐CoV‐2 proteins in scalable assays will be crucial for the success of a large‐scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme‐linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response. METHODS: Blood samples were collected from 52 individuals with a previous laboratory‐confirmed SARS‐CoV‐2 infection. These were assayed for SARS‐CoV‐2 nAbs by microneutralisation and pseudo‐type assays and for antibodies by four different ELISAs. Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels. RESULTS: All samples contained SARS‐CoV‐2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun immunoglobulin G (IgG) reactivity (Spearman Rho correlation coefficient 0.88; p < 0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). DISCUSSION: Robust associations between nAb titres and reactivity in several ELISA‐based antibody tests demonstrate their possible utility for scaled‐up production of convalescent plasma containing potentially therapeutic levels of anti‐SARS‐CoV‐2 nAbs.

UKRI/NIHR COVID-19 Rapid Response Grant (COV19-RECPLA) is likely to require high titres of neutralising antibody (nAb) in convalescent donations.

Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of a large-scale collection. We assessed whether neutralising antibody titres correlated with reactivity in a range of enzyme-linked immunosorbent assays (ELISA) targeting the spike (S) protein, the main target for human immune response.

Methods: Blood samples were collected from 52 individuals with a previous laboratoryconfirmed SARS-CoV-2 infection. These were assayed for SARS-CoV-2 nAbs by microneutralisation and pseudo-type assays and for antibodies by four different ELISAs.

Receiver operating characteristic (ROC) analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high nAb levels. ing Italy, Iran, Spain and France. 1 Subsequently, this virus was classified as SARS coronavirus 2 (SARS-CoV-2) within the genus Betacoronavirus 2 and its associated disease termed COVID-19. Mortality due to COVID-19 is as high as 50% for patients admitted to intensive care units. 3 The first imported cases of SARS-CoV-2 were identified in the United Kingdom at the end of January 2020, and local transmission within the United Kingdom became evident 1 month later. As of 1st

May 2020, a total of 182 260 cases and 28 131 deaths have been reported, and the numbers are predicted to continue to rise in this first pandemic wave. Currently, there are no approved specific antivirals targeting the novel virus, and convalescent plasma therapy (CPT) has been suggested as an immediately available therapy. A systematic review and retrospective meta-analysis, including 699 treated patients with SARS-CoV-1 infection or severe influenza and 568 untreated controls, demonstrated a statistically significant reduction in mortality and in the pooled odds of mortality following treatment, compared with placebo or no therapy (odds ratio 0.25; 95% CI: 0.14-0.45). 4 Convalescent plasma may be an effective treatment for COVID-19, with success linked to levels of neutralising antibody present in plasma, which reduce viral replication and increase viral clearance. 5, 6 Virus-specific neutralising antibodies play a key role in viral clearance.

The spike (S) protein is responsible for the SARS-CoV-2 attachment and entry to the target cells via the ACE-2 receptor, and neutralising antibodies recognising the receptor-binding domain (RBD) on the S protein have been shown to block viral entry. 7 Antibodies against other domains of S protein or possibly even against other proteins may contribute to the functional neutralisation of the virus.

Neutralising antibodies are known to be detectable in patients approximately 10-15 days after the onset of SARS-CoV-2 infection, 8 but this antibody response continues to mature for at least 3 weeks 9 and potentially longer.

The issue of the potential toxicity of convalescent plasma via antibody-dependent enhancement (ADE) also needs to be addressed carefully. It has been shown to occur when nonneutralising or heterotypic antibodies facilitate viral entry into host cells and enhance viral infectivity. 10 It is likely to occur when antibody levels or specificities do not permit neutralisation. 11 For these reasons, it is important to determine neutralising antibody titres in donated plasma, as well as a practical cut-off titre level, to evaluate not only its safety but also its effectiveness for convalescent plasma transfusion.

Neutralising antibody levels can either be determined directly using native or pseudo-type virus in cellular bioassays or be estimated by ELISA if there is an adequate correlation between neutralising antibody titre and ELISA reactivity. Neutralising antibody titre can be detected and quantified in a microneutralisation assay format in which samples are assayed for their ability to block infection of cells by SARS-CoV-2. Similarly, a pseudo-type assay can be used to measure neutralising antibody levels using a virus construct containing SARS-CoV-2 S protein in the surface of a luciferase tagged vesicular stomatitis virus or lentivirus viral vector. 12, 13 Both types of assays use suitably characterised target cells.

Although a limitation of microneutralisation assays using live virus is the necessity to undertake work at biosafety level (BSL)-3 laboratory, a pseudo-type assay is more suitable for high-throughput screening of convalescent plasma donors as it can be performed at a BSL-2 facility.

In the current study, we have first determined the neutralising antibody levels in our convalescent plasma donors and estimated a cut-off to be used in clinical trials. Second, we have also assessed whether there is a correlation between neutralisation antibody titres (measured either using microneutralisation or pseudo-type assay) and ELISA reactivity using a variety of assays formats including cell lysate, in-house assays and two commercial ELISAs. Identification of a suitable high-throughput assay is required urgently to support scaling up convalescent plasma production and to support the comparison of data between countries.

We initiated the collection of convalescent plasma using the established infrastructure and standard UK donor selection guidelines during March 2020; serum and EDTA blood samples were collected from individuals with a previous laboratory-confirmed SARS-CoV-2 infection at least 28 days after the resolution of their symptoms.

These donor samples were submitted to Public Health England and tested initially for SARS-CoV-2 RNA by in-house reverse transcription polymerase chain reaction assay, 14 as well as for SARS-CoV-2 antibodies using a native virus antigen ELISA and microneutralisation assays, both based on the UK prototype strain (GISAID accession number EPI/ISL/407073), and the samples were subsequently subjected to testing by pseudo-type neutralisation assay and trimeric S ELISA. Basic donor information including age, gender and virology testing data were collected.

Signed consent was obtained from each donor at the time of donation. Donors consent to the NHS blood and transplant holding information about them, including their health, attendances and donations, and using their information for the purposes explained in the donor welcome booklet and data protection leaflet, which donors are asked to read at the time of donation. This includes using data for the purposes of clinical audit to assess and improve the service and for research, specifically to improve our knowledge of the donor population.

Native virus antigen ELISA was modified from a previously described MERS-CoV assay. 15 Serial dilutions of convalescent plasma samples were added to microplates containing the bound detergent-extracted lysates of SARS-CoV-2 (isolate England/02/2020)-infected Vero E6 cells and uninfected cells. The reactivity was determined using a chemiluminescent substrate labelled secondary antibody. Virus lysates contain a mixture of viral proteins expressed in Vero E6 cells, including viral nucleocapsid and S proteins, and these proteins are presented in the same structure as the native virus infecting the host.

ELISA index value was defined as the difference between infected and uninfected cell reactivity expressed relative to control calibrator serum.

SARS-CoV-2 (isolate England/02/2020)-specific neutralising antibody levels were measured using a modification of the World Health Organization (WHO) influenza microneutralisation methodology. 16 Briefly, the virus was incubated with a serial dilution of convalescent plasma obtained from recovered patients, after which a suspension of VeroE6 cells was added. After 22 h, cells were fixed, and in-cell SARS-CoV-2 nucleoprotein (NP) expression was determined by ELISA. The virusneutralising antibody titre was determined as the serum concentration that inhibited 50% of SARS-CoV-2 NP expression. All work was undertaken in a BSL-3 laboratory.

Antibodies to the trimeric S (based on YP009724390.1) protein were detected by ELISA as previously described, using 2% skimmed milk in phosphate buffered saline as a blocking agent and alkaline phosphatase-conjugated anti-human IgG (A95455; Sigma) at 1:10 000 dilution. 12 Optical densities (ODs) were measured at 405 nm.

A lentivirus-based SARS-CoV-2 pseudovirus particle was constructed displaying the full S protein on the surface of pseudoparticle as previously described (accession number: YP009724390.1). 12 Neutralising antibody titres were measured by the reduction in luciferase gene expression after 72 h incubation of HEK 293T ACE2-transfected cells at 37 C. The 50% inhibitory dilution (IC 50 ) was defined as the plasma dilution at which the relative light units (RLUs) were reduced by 50% compared with the virus control wells after subtraction of the background RLUs in the groups with cells only.

EUROimmun assay is based on the S1 protein and Fortress assay on the RBD of S protein. These assays were performed according to the manufacturer's recommendation (EUROimmun, PerkinElmer, London, UK and Fortress Diagnostics, Belfast, Northern Ireland).

Associations between test assays were compared using Pearson correlation coefficients and the non-parametric Spearman's rank correlation. p-Values were derived using Student's t test for correlations and Pearson correlation coefficient under the null hypothesis that the correlation was zero. The sensitivity and specificity were calculated to assess the performance of the different assays in classifying the level F I G U R E 1 Comparison of neutralising antibody titres with reactivity in other assays. Comparison of neutralising antibody titres of the 52 test samples in the virus neutralisation assay with those of the pseudo-type assay and reactivities in enzyme immunoassay (EIAs). In all graphs, samples were ordered by virus-neutralising antibody titres. The following assay cut-off values were used: 0.049 for trimeric spike EIA, 1.0 for Fortress EIA and 1.1 for EUROimmun [Color figure can be viewed at wileyonlinelibrary.com] of neutralising antibody titres obtained by microneutralisation assay using live SARS-CoV-2 virus. Exact binomial confidence intervals were used to derive confidence intervals.

The initial assessment included samples from 52 recovered patients who would qualify as donors of convalescent plasma for clinical trials.

They were all males (to avoid the need for additional human leukocyte antigen and human neutrophil antigen antibody testing that was not available at the required scale at the time of the study) and at least 28 days from the recovery after laboratory-confirmed SARS-CoV-2 infection. They were sampled during the first 2 weeks of April, implying that their illness began at the beginning of March. Therefore, they would all have been hospitalised as a part of the containment strategy. However, no data on the severity of their infection are currently available. EDTA and serum samples were obtained from each individual, and a whole-blood donation was collected from 10. All samples were submitted to Public Health England Colindale, and available F I G U R E 2 Correlations between neutralising and pseudo-type antibody titres and reactivities in EIAs. Scatter plots of neutralising antibody titres of test samples in the virus neutralisation assay with those of the pseudo-type assay and reactivities in EIAs. A line of best fit was estimated by linear regression using log-transformed values for the virus and pseudo-type neutralising antibody assays and the EUROimmun EIA. T A B L E 1 Threshold values for optimal sensitivity and specificity of EUROimmun and pseudo-type neutralisation assays by ROC analysis In order to support the scaling up the convalescent plasma pro- As only a small number of samples from preselected convalescent plasma donors have been tested so far, which is a limitation of this study, we propose that several assay formats should be employed in a larger group of donors to validate these findings before the scaling up can be finalised. For practical and economic reasons, we decided to extend neutralising antibody testing up to 300 samples and then finalise analysis. Nevertheless, the results provide guidance for the many convalescent plasma programmes in progress around the world.

Neutralising antibody levels are partly dependent on the timing of collection relative to the recovery from infection. Seroconversion following SARS-CoV-2 infection has been observed between 8 and 21 days after the onset of symptoms, 9, [19] [20] [21] and higher levels of antibodies have been determined in plasma collected at least 14 days after the symptom resolution. 5 It is likely that the antibody maturation continues for longer as demonstrated for other viruses, and hence, the collection point of 28 days after recovery has been chosen here.

This maximises the chances of collecting the most clinically effective donations. However, it is still unclear how long neutralising antibody levels are maintained, and hence, repeat testing will be performed at every donation.

Higher neutralising SARS-CoV-2 antibody levels have been associated with older age and a worse clinical outcome, 8, 21 although good neutralising antibody levels have also been measured in individual patients with milder infections. 22, 23 The monitoring of neutralising antibody levels in different patient groups (including females not included in this study) and over time is required and will inform future screening strategies.

In conclusion, here, we have demonstrated a correlation between the neutralising antibody level and antibody reactivity measured by ELISA, which will allow scaling up of the convalescent plasma production. However, continuous monitoring of assay performance, antibody decay and adaptation of selection strategies will be required in order to deliver the best clinical outcomes for patients receiving neutralising SARS-CoV-2 antibodies through CPT.

We thank all the donors who have kindly donated convalescent plasma. We are grateful for everybody within the NHS Blood and 

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The authors have no competing interests.

Heli Harvala: designed the study; coordinated the testing and col-