Convalescent plasma for COVID‐19: Donor demographic factors associated high neutralising antibody titres

1. INTRODUCTION

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) was first recorded in China at the end of 2019 and has since continued to spread throughout the world. While many eagerly await the coronavirus vaccinations as the main pandemic exit strategy, the search for effective prophylactic and supportive therapies to treat vaccine non‐responders continues. ¹ Convalescent plasma containing high levels of neutralising antibodies to SARS‐CoV‐2 is a potential means to reduce morbidity and mortality. Although recent meta‐analysis including 33 convalescent plasma trials with 16 477 patients with COVID‐19 demonstrated that convalescent plasma treatment did not decrease all‐cause mortality, ² , ³ it may be beneficial if given in early stages of infection ⁴ , ⁵ , ⁶ , ⁷ or used to treat those unable to mount an effective immune response. ³ , ⁸ , ⁹ , ¹⁰ Although immunisations programmes are advancing very fast, rapid spread of new SARS‐CoV‐2 variants with several mutations in the spike (S) glycoprotein that may show increased resistance to neutralisation have raised concerns ¹¹ , ¹² , ¹³ and might support future use for convalescent plasma.

We have previously demonstrated a correlation between virus neutralising antibody titres and antibody reactivity in commercial EUROimmun ELISA IgG tests in convalescent plasma collected from individuals with confirmed or suspected SARS‐CoV‐2 infection at least 28 days after the resolution of their symptoms. ¹⁴ , ¹⁵ Based on that, we adopted a strategy where all donations with a signal to cut‐off (S/CO) ratio of 6.0 or higher in the EUROimmun assay were released for clinical use without further virus neutralisation testing. ¹⁴ Replacement of live virus neutralisation testing performed externally with in‐house EUROimmun testing has allowed testing of a large number of donations in a very short timeframe. All convalescent plasma donations are tested for SARS‐CoV‐2 IgG antibodies by EUROimmun assay at NHS Blood and Transplant testing laboratories in Filton and Manchester, and results transferred directly into our donor management database.

A better understanding of the predictors of high antibody levels is important for improving access to high‐quality therapeutic plasma. It has been previously demonstrated that high neutralising antibody levels correlated with male sex, older age, and severity of SARS‐CoV‐2 infection, ¹⁶ , ¹⁷ albeit derived from studies with small sample sizes (<500). To obtain more definitive data on predictive factors for high neutralising antibody levels, we have evaluated donor demographic and clinical factors associated with high antibody levels based on the EUROimmun IgG result in a large cross‐sectional study of 29 585 donors in England in 2020.

2. METHODS

3. RESULTS

A total of 29 585 convalescent plasma donors were included in our study; they all either donated plasma or were sampled in England between 22nd April and 16th December 2020. Most of them were male (23 024; 78%), with white ethnic background (24 598; 83%), had either blood group A (12,259; 42%) or O (12,741; 43%), donated in the London region (9759; 33%), were new blood donors (23 210; 78%) and had not been tested for SARS‐CoV‐2 (15 266; 52%; Table 1). Only a small proportion of our donors had been hospitalised with SARS‐CoV‐2 infection (1039; 3.5%). Overall, 4978 (17%) convalescent plasma donors exhibited high SARS‐CoV‐2 antibody levels as defined by a EUROimmun S/CO ratio of 6 or higher. A high proportion of Asian and Black ethnicity donors showed high levels of antibodies (39% and 30%, respectively), but the highest proportion of donors with high antibody levels were seen among those who had been hospitalised with SARS‐CoV‐2 infection (64%). The proportion of donors with EUROimmun S/CO ratio of 6 or higher by ‘time since project began’ and ‘duration between SARS‐CoV‐2 detection and donation’ are summarised in Figures 1 and 2 respectively.

Multivariable modelling was used to identify variables associated with high antibody levels whilst adjusting for the effect of all the factors we tested found to be influential. Data from 20 795 donors was used for model development, and from 8790 donors for model validation. The final model was based on 20 073 donors when excluding donors with missing blood group and social deprivation data; similarly, 8511 donors were included in validation set. All the factors considered in the analyses were found to significantly influence the probability of high SARS‐CoV‐2 antibody levels (defined as EUROimmun S/CO ratio 6.0 or higher) at the 10% significance level with the exception of time between SARS‐CoV‐2 detection and donation (Table 2). Note that this factor could only be tested for donors that had been tested for SARS‐CoV‐2 and a test date was reported (n = 14 319). Four two‐way interaction terms were found to be significant and retained in the model: gender and test group (p = 0.002), age and test group (p = 0.02), age and gender (p = 0.04), ethnic group and social deprivation indicator (p = 0.04). Two further two‐way interaction terms were found to be significant (previous blood donor and gender, previous blood donor and test group) but the clinical interpretation was thought to be questionable and so these terms were excluded from the model. One final interaction term, between social deprivation and age, was found to be significant but was kept out of the model firstly because its inclusion would overcomplicate the interpretability of the model (age was already involved in two other significant interaction terms) and secondly because the p‐value for an interaction term was relatively high at 0.09 and hence considered less important than the other interaction terms involving age.

In our multivariable analysis, blood group A donors (OR 1.14, 95% CI 1.04–1.24) were found to have a significantly higher probability of high SARS‐CoV‐2 antibody levels than blood group O donors, whereas donors with AB blood group (OR 0.77, 95% CI 0.62–0.97) were more likely to have lower antibody levels compared to blood group O donors (Table 2).

The influences of age, gender, and test group on the probability that a donor would have high SARS‐CoV‐2 antibody levels were interpreted collectively due to significant interactions identified between these variables (Table 2). Male donors who had tested positive for SARS‐CoV‐2 but had not been hospitalised were more likely to have high antibody levels than those without a SARS‐CoV‐2 positive test across all age groups (Figure 3). Male donors with a laboratory confirmed SARS‐CoV‐2 diagnosis who were hospitalised were even more likely to have high antibody levels than those who had not been tested or hospitalised, and this impact was pronounced by age: those male donors who were 65 years of age or over, were tested positive for SARS‐CoV‐2 and hospitalised were most likely to harbour high antibody levels (OR 37.07, 95% CI 8.58–160.14). A similar relationship between age, gender, laboratory diagnosis and hospitalisation status were found for females, but the odds of high SARS‐CoV‐2 antibody levels were generally lower than for males (Figure 4). However, it is important to note that some of these differences were based on very small numbers of donors. For example, although female donors aged over 65 years who had been hospitalised with COVID‐19 were more likely to have high SARS‐CoV‐2 antibody levels than younger females (OR 17.42, 95% CI 4.02–75.50), that group only included seven members.

Similarly, the effects of donor ethnicity and social deprivation indicator were assessed together due to a significant interaction identified between these two factors (Table 2). White donors were less likely to have high SARS‐CoV‐2 antibody levels than donors of other ethnic backgrounds (Figure 5). Although it is possible that this could partially reflect differences in ability or willingness to access SARS‐CoV‐2 testing, a two‐way interaction term between test group and ethnicity was not found to be significant (p = 0.29) and is not included in the final model. However, the effect of ethnic group differed depending on social deprivation indicator. While donors of Asian origin were found to have the highest odds of high antibody levels across all social deprivation groups, the odds compared to white donors were highest in the more affluent areas compared to the more deprived areas (OR: 4.33, 95% CI 3.29–5.69 versus OR: 2.05, 95% CI 1.29–3.26). Interestingly, black donors were more likely to have high SARS‐CoV‐2 antibody levels compared to white donors but only when considering the most affluent areas (OR 3.80, 95% CI 1.58–9.12). This latter result was not due to lack of statistical power.

We also assessed the significant (p < 0.0001) non‐linear effect of timing of donation (days since the project began) on the probability of high SARS‐CoV‐2 antibody levels (Figure 6). The odds of donors having high antibody levels appears to have decreased over time since the beginning of the project. However, the rate of decrease slowed down around mid‐October (day 170) co‐inciding with the increase in reported SARS‐CoV‐2 cases in the United Kingdom.

As the Hosmer and Lemeshow C statistic was >0.7, the model was considered to exhibit acceptable predictive ability. Residual analysis highlighted one donor that influenced the age and test group interaction parameter estimate however this donor was kept in the dataset as it was a genuine observation that led to more conservative results. Some variables with smaller numbers of observations were, by definition, slightly more susceptible to influential values but there were no observations that posed a particular concern.

4. DISCUSSION

This is a large convalescent plasma donor cohort study to date reflecting a commitment of time, resources and generosity from NHS staff and almost 30 000 donors who took part in the convalescent plasma programme in England. To investigate the trends in demographic and clinical factors that influence the probability of individuals with previous SARS‐CoV‐2 infection to produce a good antibody response in such a large dataset has allowed us to draw robust conclusions from these data.

We showed that older donors, male donors, and donors previously hospitalised with SARS‐CoV‐2 infection were generally much more likely to have high levels of antibodies. This was consistent with previous findings, based on neutralising antibody testing of a smaller set of donors. ¹⁶ It should be noted that during the course of the study, there was a substantial drive to recruit male donors because the preliminary analysis suggested a greater probability of high antibody levels compared to females. ¹⁶ Overall, 78% of donors were male. Female donors were generally only recruited if they had confirmed SARS‐CoV‐2 and thus thought to have increased probability of high antibody levels. Sex differences in COVID‐19 severity were first noted in China; hospital admissions and mortality were higher among males than females. ¹⁸ , ¹⁹ , ²⁰ Further male bias in COVID‐19 mortality has been reported by 37 of the 38 countries providing sex ‐disaggregated data. ²¹ It has been proposed that these differences observed between males and females in response to SARS‐CoV‐2 infection are likely due to differences in their innate and adaptive immune responses driven by biological sex, including sex chromosomes and sex steroids (reviewed by Scully et al). ²¹ Similarly, differences in mortality between males and females were previously investigated in animal models of SARS‐CoV infection, and these were attributed to steroid hormones. ²²

With this dataset we were also able to demonstrate that donors from an Asian background had higher likelihood of having high SARS‐CoV‐2 antibody levels compared to white donors; although this disparity was enhanced in affluent areas, it was also seen in deprived areas. In contrast, the likelihood of having high SARS‐CoV‐2 antibody levels was increased only among those black donors residing in affluent areas. Data on the disproportionate effect of SARS‐CoV‐2 infection on black, Asian, and other minority ethnic people and possible reasons underlying that has been recently reviewed. ²³ The results suggest that people of black and Asian ethnic groups are more likely to be diagnosed with COVID‐19. People from Asian and black ethnic groups have also been shown to be more likely hospitalised with SARS‐CoV‐2 infection when compared to those with white ethnicity (threefold and twofold, respectively). Furthermore, the evidence emerging from the United Kingdom suggests that those of black or Asian ethnicity are around twice as likely to die from SARS‐CoV‐2 infection than those of white British ethnicity. ²⁴ It has been proposed that black and Asian people are more likely to work in occupations with a higher risk of SARS‐CoV‐2 exposure or due to overcrowded housing, and might also delay seeking medical care when needed due to their negative past experiences with healthcare. ²³ Although these cultural differences may impact the disease risk, it has also been shown recently that poverty is another independent risk factor for COVID‐19 related mortality. ²⁵ Based on our results, blood donors with Asian background, residing in deprived areas had higher likelihood of high antibody levels than white donors, likely demonstrating increased exposure to SARS‐CoV‐2 infection. This could reflect the previous findings where the proportion of black household lone parents is higher when compared to other groups, making them possibly more isolated and hence less likely to get infected. Similarly, self‐employment rates are highest among Pakistanis and Bangladeshis ²⁶ potentially leading to their more frequent exposure to SARS‐CoV‐2 infection if it has not been possible to isolate due to financial pressure.

Donors with blood group A were more likely to have high SARS‐CoV‐2 antibody levels than those with blood group O, and those with blood group AB were significantly less likely to harbour high antibody levels compared with blood group O donors. Based on a previous meta‐analysis covering over 50 000 patients, individuals with blood group A have a substantially higher risk of SARS‐CoV‐2 infection and blood group A has also been associated with an increased risk of COVID‐19 mortality. ²⁷ Furthermore, blood group O has shown to be a protective factor reducing the risk of SARS‐CoV‐2 infection. It has been speculated previously whether this association might relate to the presence of anti‐A antibodies via their ability to block the interaction of SARS‐CoV‐2 with the ACE receptor, making those with blood group A without these antibodies more vulnerable to SARS‐CoV‐2 infection than those with blood group O. ²⁸ , ²⁹ , ³⁰ This protective effect of anti‐A antibodies was not seen among those with blood group AB in our study, indicating that this is likely a multifactorial issue, perhaps also involving other ABO‐associated factors such as A and B antigens, coagulation factors and interleukin levels. ³¹

It is difficult to explain why the odds of blood donors having high antibody levels decreased during the study period; the odds of high antibody levels on 16 December were 83% lower than on 22 April 2020. Although it is currently unknown whether individuals' low antibody titres will boost their immune responses upon re‐exposure or because of SARS‐CoV‐2 immunisation, this has been suggested based on experimental infection of ferrets. ³² As a smaller number of SARS‐CoV‐2 infections were diagnosed in the UK during the first outbreak between March and May than during the second peak in the Autumn, it might have been anticipated that the number of donors with high SARS‐CoV‐2 antibody levels would have increased during the study period. However, in the beginning of the pandemic SARS‐CoV‐2 testing was limited to those with severe symptoms, whereas during the subsequent months, testing increased to include those with milder symptoms and hence our convalescent plasma population likely contained more donors with a previous SARS‐CoV‐2 diagnosis but with mild symptoms only. It might also be that the decrease in proportion of donors with high antibody levels over the study period was reflected by a general rapid increase in convalescent plasma collections, likely containing more donors from lower‐priority groups expected to have lower likelihood to have high antibody levels (i.e. women, younger individuals and those untested). Interestingly, most convalescent plasma donors had not donated previously and were hence newly recruited (78%). This is in contrast to our usual (blood) donor demographics where only 10% of donations are given by new donors (accounting for 17% of all donors). ³³ Our results also show that donors who had previously donated blood had 16% lower odds of high SARS‐CoV‐2 antibody levels compared to those who had not previously given blood, which is likely explained by the targeted recruitment of new convalescent plasma donors, focusing on males with previous laboratory confirmed SARS‐CoV‐2 infection and calling on existing blood donors to ‘sign up’ as a donor if they felt they had suffered from COVID‐19.

Reassuringly for the convalescent plasma programmes and those having been infected with SARS‐CoV‐2, the interval between a laboratory confirmed SARS‐CoV‐2 diagnosis and plasma donation was not found to be significantly associated with the likelihood of them having high antibody levels for the range of durations in our cohort (up to 327 days), indicating that in some donors high SARS‐CoV‐2 antibody levels remain over time. Decreases in SARS‐CoV‐2 neutralising antibody levels have been demonstrated in some ³⁴ , ³⁵ , ³⁶ , ³⁷ but not all previous studies. ³⁸ One of these studies demonstrated a trend towards lower antibody levels with increased time between symptoms and blood sampling, and a decline of neutralising antibody titres over time based on repeat testing of the same donors. ³⁷ The difference between our results and their findings cannot be explained by methodology as Euroimmun IgG assay was utilised in both studies. Furthermore, although both studies focused on convalescent plasma donors, it is important to note that the donor populations were very different. Our study included a higher proportion of males than the study by Boonyaratanakornkit et al ³⁷ (78% vs. 48%), a smaller proportion of donors had been hospitalised (3% vs. 10.8%) and a smaller proportion of them had high antibody levels (17% vs. 60%). Whether these highlighted differences in our donor populations could be associated with different levels of immune memory needs further investigation.

Although our study is based on a large dataset, it also has some limitations. It is important to note that when we consider the effect of age in the model, all donors over 65 years of age must have been previous blood donors and might explain why there are fewer high titre donors in that age group than among those aged between 55 and 64 years. Furthermore, our cohort is composed of eligible blood donors only and all of them had to pass the medical assessment. Approximately 60–70% of female and 30–40% of male first‐time donors attending a donation session were actually deferred without testing. For these reasons, our cohort is selective and relates to healthy people eligible to donate convalescent plasma only.

Due to the extensive size of our dataset, we have been able to perform detailed model checking and validation exercises leading us to conclude that our model provides a satisfactory fit to the data and has acceptable predictive ability. With this large dataset we have shown that not only older male donors who were hospitalised with SARS‐CoV‐2 infection but also donors with an Asian background and donors with a black background residing in affluent areas demonstrate higher likelihood of having high SARS‐CoV‐2 antibody levels. In a time of great uncertainty, these results take us one step closer to understanding the antibody response to COVID‐19 and help us to target convalescent plasma collections further.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

All authors discussed and planned the study, Jennifer Mehew performed the multivariable analysis and drafted the first version of manuscript together with Heli Harvala. All authors critically reviewed the manuscript and accepted the last version of it.

Supporting information

Mehew J, Johnson R, Roberts D, Griffiths A, Harvala H. Convalescent plasma for COVID‐19: Donor demographic factors associated high neutralising antibody titres. Transfusion Medicine. 2022;1‐11. doi: 10.1111/tme.12868