Endemic Human Coronavirus Antibody Levels Are Unchanged after Convalescent or Control Plasma Transfusion for Early Outpatient COVID-19 Treatment

ABSTRACT The impact of preexisting antibodies to the four endemic human coronaviruses (ehCoV) (229E, OC43, NL63, and HKU1) on severe (hospitalization) coronavirus disease 2019 (COVID-19) outcomes has been described in small cohorts. Many studies have measured ehCoV 229E, OC43, NL63, and HKU1 antibody levels weeks after recovery rather than in the first weeks of illness, which is more relevant to early hospitalizations. Antibody levels to the spike protein of the four coronaviruses (229E, OC43, NL63, and HKU1), as well as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were measured both before and immediately after convalescent or control plasma transfusion in 51 participants who were hospitalized and 250 who were not hospitalized, as well as in 71 convalescent and 50 control plasma donors as a subset from a completed randomized controlled trial. In COVID-19 convalescent plasma donors, the ehCoV spike antibodies were 1.2 to 2 times greater than the control donor unit levels, while donor COVID-19 convalescent plasma (CCP) SARS-CoV-2 spike antibodies were more than 600 times the control plasma units. Plasma transfusion, whether COVID-19 convalescent or control, did not alter the post-transfusion antibody levels for the endemic human coronaviruses (229E, OC43, NL63, and HKU1) in those hospitalized and not hospitalized, despite the 1.2- to 2-fold elevation in donor COVID-19 convalescent plasma. There was no influence of prior antibody levels to 229E, OC43, NL63, and HKU1 or post-transfusion antibody levels on subsequent hospitalization. These data, from a well-controlled prospective randomized clinical trial, add evidence that antibodies to ehCoV do not significantly impact COVID-19 outcomes, despite the apparent back-boosting of some ehCoV after SARS-CoV-2 infection.

IMPORTANCE The relevance of preexisting immunity to the four endemic human coronaviruses in the first week of COVID-19 illness on the outcome of COVID-19 progression stems from the high prevalence of the ehCoV and SARS-CoV-2 coronaviruses. The question has been raised of whether therapeutic convalescent plasma or control plasma containing ehCoV antibodies might alter the outcome of COVID-19 progression to hospitalization. Here, we observed that plasma transfusion did not significantly change the preexisting ehCoV antibody levels. In over 50 hospitalized participants and 250 nonhospitalized participants, ehCoV antibody levels were comparable, without statistical differences. Antibody levels were stable over the more than 12 months of the intervention trial, with individual heterogeneity similar in hospitalized and nonhospitalized participants. The ehCoV antibodies in plasma transfusion did not alter the recipient preexisting antibody levels nor hasten the COVID-19 progression to hospitalization in this clinical trial data.
The prospective association of severe COVID-19 with elevated ehCoV antibody levels at illness onset has not been investigated (12). A clinically relevant question was raised recently as to whether antibody levels to ehCoV in control plasma as opposed to saline could deleteriously influence hospital outcomes in controls during a clinical trial (13). The literature has mainly looked retrospectively for correlations of high antibody levels weeks after hospitalization, even though COVID-19 hospitalization itself raises antibody levels to both ehCoV and SARS-CoV-2 (3,4).
This substudy of a larger trial (14) focuses on the impact of preexisting ehCoV antibody immunity on the outcome of severe SARS-CoV-2 disease by measuring ehCoV antibody levels in the first week of COVID-19, rather than taking a retrospective look back weeks after recovery. Additionally, we examined ehCoV and SARS-CoV-2 antibody levels from unused CCP and nonconvalescent control donor units, as well as before and after randomized transfusion (within the first 9 days of COVID-19), in a representative sampling of participants from a subset of an interventional randomized clinical trial, which demonstrated efficacy at outpatient hospital reduction (14).
Antibodies that bind the spike proteins from 229E, NL63, OC43, HKU1, and SARS-CoV-2 were measured using the Meso Scale Diagnostics (MSD; Rockville, MD) V-PLEX COVID-19 coronavirus panel 3 IgG kit at a dilution of 1:5,000. Plates were read on a Meso QuickPlex SQ 120 instrument, and the arbitrary units (AU) were calculated using the MSD Discovery Workbench software according to the manufacturer's protocol. For the samples in the spike competition experiment, purified recombinant full-length WA-1 SARS-CoV-2 spike protein (150 mg/mL) (4) was incubated 1:1 with plasma samples for 45 min prior to antibody measurement via the MSD assay as described above. Approvals were obtained from the institutional review boards at Johns Hopkins University School of Medicine under a single IRB (IRB00247590) for all participating sites and the Department of Defense (DoD) Human Research Protection Office. All the trial participants provided written informed consent for the scope of this research.
The nonhospitalized subset (n = 250) randomly chosen for endemic human CoV antibody analysis was similar to the full study with regard to key demographic features. This subgroup had a similar percentage of female participants, with similar comorbidities (hypertension, lung disease, diabetes) and a small 4-year mean age difference (see Table S1 in the supplemental material). SARS-CoV-2 vaccination at enrollment was excluded for this substudy.
In CCP donors, the ehCoV spike antibodies were 1.2 to 2 times greater than the control donor unit levels, while the donor CCP SARS-CoV-2 spike antibodies were more than 600 times the levels of the control units (Fig. 1A). In the SARS-CoV-2 CCP participants who were seronegative for the SARS-CoV-2 receptor-binding domain (RBD) by Euroimmun assay, the SARS-CoV-2 spike antibodies increased 17-fold from pretransfusion screen to post-transfusion, while the HKU1 and OC43 antibodies showed a small insignificant increase. In separate seronegative control recipients, the ehCoV antibodies pretransfusion to posttransfusion were also unchanged (Fig. 1B, C). We also measured antibody levels pre-and post-transfusion in 50 participants who were seropositive at enrollment (Fig. S1A, B). Baseline pretransfusion SARS-CoV-2, HKU1, and OC43 antibody levels in seropositive CCP and controls were increased compared to seronegative participants, with no change post-transfusion for all viruses. In the hospitalized CCP and control plasma participants, the pretransfusion and posttransfusion geometric mean antibody levels were similar, except for SARS-CoV-2 in CCP recipients ( Fig. 2A, B). A comparison of pretransfusion antibody levels in all 51 hospitalized participants to the levels in those not hospitalized indicated no differences in ehCoV levels (Fig. 2C).
The increase in convalescent antibody levels against HKU1 and OC43 seen in this study, both of which are also betacoronaviruses, might be from cross-reactivity with elevated SARS-CoV-2 spike antibodies or induction of ehCoV-specific B cells to generate more ehCoV antibodies. We added purified SARS-CoV-2 spike at 150 mg spike/mL plasma (4) to donor unit plasma samples. SARS-CoV-2-specific antibodies were reduced 86% and HKU1 by 21%, suggesting minimal measurable cross-reactivity with HKU1 spike only (Fig. S2). Heterogeneity heat maps of antibody levels sorted by 229E indicated similar patterns among those hospitalized to those not hospitalized (Fig. S3). The donor CCP and control plasma had similar antibody levels despite regional donations (Fig. S4). Despite minimum ongoing ehCoV transmission due to social isolation measures during the trial, over time the remnant donor CCP and control plasma, as well as, in participants, the four ehCoV antibody levels, were the same over the more than 12 months of the study (Fig. S5 to S8). We observed no significant post-transfusion ehCoV antibody level changes in either the CCP or control plasma recipients to account for intervention arm differences in hospitalization from either ehCoV antibody COVID-19 cross-protection or enhancement. Many other studies measured 229E, OC43, NL63, and HKU1 antibody levels weeks after illness rather than during the first week of illness. The first week of illness had a higher predictive value on early hospitalizations, which mostly happened within a week of transfusion or within 2 weeks of illness onset.
The antibody levels of ehCoV in COVID-19 convalescent plasma were only 2-fold different between the control and convalescent plasma. The volume of distribution of the plasma was 3 to 6 L. The measured fold dilution when transfused into healthy individuals was 25-fold with a 200-mL CCP transfusion (15). When plasma with ehCoV antibodies was diluted 20-fold, it resulted in no change before or after transfusion, as shown here in over 300 paired samples, of which 51 donors were later hospitalized. Likewise, in those clinical trial recipients who were SARS-CoV-2 antibody negative or positive, there was no change in the levels before or after transfusion, but those who were seropositive for SARS CoV-2 at enrollment had HKU1 and OC43 levels that were twice as high compared to the seronegative participant ehCoV levels, which mirrored the levels seen in donor CCP compared to control plasma, with only the 2fold difference, which, again, did not change the post-transfusion antibody levels.
Measurement of the available 17 hospitalized participants in the COVID-19 convalescent plasma arm and the 34 in the control plasma arm indicated no absolute ehCoV antibody level differences between these two groups or with 250 other paired participants. Even combining all 51 hospitalized participants to compare them to the 250 participants not hospitalized at screening before transfusion showed no differences.
In this study, the EhCoV antibody levels of those hospitalized and those not hospitalized were not distinguishable during the first week of illness, at which time there exists greatest risk for impact on severe COVID-19 outcome. Thus, it is unlikely that preexisting ehCoV antibodies in plasma have any clinically relevant impact on COVID-19 outcomes.
Data availability. The data sets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. This study was funded principally by the U.S. Department of Defense's (DOD) Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense (JPEO-CBRND), in collaboration with the Defense Health Agency (DHA) (contract number W911QY2090012), with additional support from Bloomberg Philanthropies, State of Maryland, the National Institutes of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID) (3R01AI152078-01S1, K08AI156021, U54CA260491, and R01AI138897), the NIH National Cancer Institute (NCI) (U54CA260491), the NIH National Center for Advancing Translational Sciences (NCATS) (U24TR001609-S3), the Division of Intramural Research NIAID NIH, the Mental Wellness Foundation, the Moriah Fund, Octapharma, the Healthnetwork Foundation, and the Shear Family Foundation. The study sponsors did not contribute to the study design, the collection, analysis, and interpretation of data, or the decision to submit the paper for publication.
We report no competing interests to this work. A. H. Karaba reports receiving consulting fees from Roche. D. J. Sullivan reports that he is an AliquantumRx founder and board member with stock options (macrolide for malaria), works for Hemex Health malaria diagnostics consulting, and receives royalties for malaria diagnostic test control standards from Alere-all outside the submitted work.
The members of the COVID-10 Serologic Studies Consortium are as follows: Barry R.