Testing matter – SARS-CoV-2 neutralising antibody assays, variants and the optimum plasma donor

Doctors Heli Harvala and Emma Wakins.

COVID-19 has taught us much about the value of collaboration - in our laboratories, as well as in our teams and in our local communities. At a time when we were working away from one another in a physical sense, clinical and scientific staff have never worked more closely, or been under greater pressure to deliver results quickly to guide patient treatment.

Here, we summarise the findings of our studies covering some key aspects on SARS-CoV-2 – assays to identify plasma for therapeutic use in patients with COVID-19, characteristics of the ideal convalescent plasma donor, and the virological characterisation of critically ill patients with COVID-19.

Effective convalescent plasma (CVP) therapy in patients with coronavirus disease (COVID-19) requires high concentration (titres) of neutralising antibodies (nAb). One of the first studies that we completed looked at the correlation between reactivity in enzyme-linked immunosorbent assays (ELISA) available at the time and the nAb titre measured with live virus assay of the plasma (1). To support a potential increase in CVP production, it was necessary to identify a suitable high-throughput assay that could determine the nAb titre in these plasma samples and thereby identify donations for clinical use.

Blood samples collected from the first 52 donors with a previous laboratory-confirmed SARS-CoV-2 infection were included in this first collaborative study with Public Health England and the University of Oxford. The strongest correlation between nAb titres and ELISA reactivity was observed in the Euroimmun immunoglobulin (IgG) ELISA; and led to its introduction into our plasma donor screening as early as in May 2020. A nAb titre of 1:100 was agreed as a pragmatic cut-off to identify donations for clinical use, corresponding to the ratio of 9.0 in Euroimmun assay. We could now test a greater number of CVP donations using a simple standard technique and provide them for clinical use.

However, as we had only worked with a very small number of samples, we continued to assess the effectiveness of commercial assays in a much larger study with Public Health England (2).This study included further 436 CPV samples collected in England during April and May 2020. It confirmed that the commercial Euroimmun ELISA was a suitable assay and allowed us to refine our Euroimmun threshold from 9 to 6 equalling a nAb titre of at least 1:100. We then focussed our attention on identifying the optimum CVP donor.

In the study by Mehew et al. (2020), we investigated the clinical and demographic factors that are likely to be linked to high nAb titres in CVP donors (3). We analysed the data on 275 donors who had a previous PCR-confirmed SARS-CoV-2 infection and had detectable nAb against SARS-CoV-2 (titre range 1:12 – 1:2560). We subjected the results to a multi-variable analysis, to look for correlations between nAb titre and sex, age, blood group, ethnicity, location of donation, hospitalisation, social deprivation and the time between diagnosis and plasma donation. Levels of nAb were higher in men and in older donors, higher in donors who had been hospitalised with COVID-19 and higher in those who donated in Edgware or Manchester centres. Further analysis is required to explain the differences between donations given in different parts of England. The nAb levels decreased as the time between diagnosis and donation increased. The results were consistent with the findings of US studies that demonstrated an association between nAb titre and male, older donors who required hospitalisation.

Ethnicity wasn’t a significant factor in the analysis but some of the ethnicity data wasn't available. We couldn't draw firm conclusions on the influence of blood group, but this has been looked at in other countries and is worthy of future investigation. These data helped us to focus our convalescent plasma collection to older males who had been hospitalised with COVID-19.

Having identified the characteristics of the ideal donor, we wanted to see whether the virological and serological features of the patient affected the efficacy of treatment with nAb against SARS-CoV-2 (4). This study included over 1000 patients enrolled in the REMAP-CAP trial in the UK from May 2020 to January 2021. All these patients had a laboratory-confirmed diagnosis of SARS-CoV-2 infection with connected severe pneumonia requiring ICU admission.

We found variability in pre-treatment viral loads and serological status, along with increased detection of the UK B.1.1.7 strain (subsequently named as Alpha variant) – all important factors that influence the effectiveness of CVP therapy. Treatment with nAb could be less relevant in patients who have already seroconverted, so it is important to identify the stage of infection. Virus strain can also affect patient outcomes. If variants are less susceptible to nAb, then this will reduce the efficacy of CVP.

In our final paper of the series, we return to the key theme of collaboration as we report on efforts to standardise nAb testing across the UK and Europe, to allow us to compare the results of different convalescent plasma and vaccine studies (5). 12 laboratories in the UK and 8 other European countries were provided with CVP samples of varying titres. The data demonstrated that we can get comparable results from different laboratories providing the use of international standards – this is extremely important for large clinical trials assessing clinical efficacy. There were lower antibody levels in samples of the variants. This fits with what we already know and crucially, it highlights the importance of monitoring the emergence of variants and the importance of sampling CVP recipients. It also demonstrates that we need an international standard for every variant of concern.

Key learning points

Collaboration is vital but there are still challenges around data sharing agreements. We are learning more all the time and we couldn't have made progress without the help of plasma donors. Researchers should make use of existing arrangements and protocols – time is of the essence. Above all, clinicians and scientists need to prepare for the threat of emerging infections and be ready to respond to the challenge!

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R&D Blog Co-Authors

Emma Watkins (PA to Dr. Heli Harvala) and Dr. Heli Harvala (NHSBT Principal Investigator)