key: cord-0934054-a6au5uvz
authors: Leon, Judith; Merrill, Anna E.; Rogers, Kai; Kurt, Julie; Dempewolf, Spencer; Ehlers, Alexandra; Jackson, J. Brooks; Knudson, C. Michael
title: SARS-CoV-2 antibody changes in patients receiving COVID-19 convalescent plasma from normal and vaccinated donors
date: 2021-11-23
journal: Transfus Apher Sci
DOI: 10.1016/j.transci.2021.103326
sha: b54b54ee7153225545da4cea68b1a2947d02b66e
doc_id: 934054
cord_uid: a6au5uvz

Vaccination has been shown to stimulate remarkably high antibody levels in donors who have recovered from COVID-19. Our objective was to measure patient antibody levels before and after transfusion with COVID-19 Convalescent Plasma (CCP) and compare the antibody levels following transfusion of CCP from vaccinated and nonvaccinated donors. Plasma samples before and after transfusion were obtained from 25 recipients of CCP and COVID-19 antibody levels measured. Factors that effect changes in antibody levels were examined. In the 21 patients who received CCP from nonvaccinated donors, modest increases in antibody levels were observed. Patients who received two units were more likely to seroconvert than those receiving just one unit. The strongest predictor of changes in patient antibody level was the CCP dose, calculated by the unit volume multiplied by the donor antibody level. Using patient plasma volume and donor antibody level, the post-transfusion antibody level could be predicted with reasonable accuracy(R 2 > 0.90). In contrast, the 4 patients who received CCP from vaccinated donors all had dramatic increases in antibody levels following transfusion of a single unit. In this subset of recipients, antibody levels observed after transfusion of CCP were comparable to those seen in donors who had fully recovered from COVID-19. If available, CCP from vaccinated donors with very high antibody levels should be used. One unit of CCP from vaccinated donors increases patient antibody levels much more than 1 or 2 units of CCP from unvaccinated donors.

following transfusion of a single unit. In this subset of recipients, antibody levels observed after transfusion of CCP were comparable to those seen in donors who had fully recovered from COVID-19. If available, CCP from vaccinated donors with very high antibody levels should be used. One unit of CCP from vaccinated donors increases patient antibody levels much more than 1 or 2 units of CCP from unvaccinated donors.

Keywords: COVID-19; SARS-CoV-2, COVID-19 convalescent plasma (CCP), vaccinated donors

The emergence of SARS-CoV-2, the causative agent of COVID-19, has resulted in intense efforts to identify new and effective treatments. The lack of clinically validated anti-viral therapies against coronaviruses led to the broad utilization of COVID-19 convalescent plasma (CCP) obtained from survivors of COVID-19 to treat patients with active disease [1] [2] [3] . While the mechanism of action of CCP is uncertain, the most prevalent hypothesis is that CCP contains neutralizing antibodies that limit viral spread and replication [4] . Multiple reports describe the rationale for this therapy and several studies provide some evidence of efficacy [5] [6] [7] [8] [9] [10] . However, other trials have failed to show the benefit of CCP in hospitalized patients [11] and meta-analyses to date have drawn equivocal conclusions about the efficacy of CCP [12] [13] [14] . The closing of both the REMAP-CAP trial (NCT02735707) and the RECOVERY trial (NCT04381936), halted due to futility, have dampened enthusiasm for CCP. However, CCP continues to be used for select patient populations. One common limitation in these trials is that patient SARS-CoV-2 antibody levels were not measured before enrollment, with retrospective antibody testing identifying many patients who were seropositive before treatment [11] . Antibody responses following CCP transfusion has not been broadly studied and when it has been studied the changes in antibody levels are often modest. While not a randomized trial, results from the expanded access protocol in the US (NCT04338360) demonstrate that hospitalized patients receiving high titer CCP had improved survival when compared to the low titer group [15] . Furthermore, recent reports have demonstrated that CCP donors who are vaccinated have dramatically higher spike-specific antibody levels with high neutralization titers than those that experience a natural infection [16, 17] . Given this information, one could hypothesize that that CCP from vaccinated donors could be a more efficacious product than CCP from nonvaccinated donors.

In the early days of the current pandemic groups around the world rushed to utilize convalescent plasma obtained from COVID-19 survivors without clear guidelines for the selection of the best candidates for donation of CCP or identifying clinical parameters for patients most likely to benefit from CCP. This approach did not allow for early phase clinical trials that would look at the pharmacokinetics of the intervention in question and help with the dosing and timing of CCP as trials were designed. As a CCP donor program was established in our hospital-based donor center, nearly all the CCP used at our hospital came from donors with known antibody levels. To begin to identify what makes some CCP units more effective than others, an IRB-approved protocol to collect and store plasma on CCP recipients before and after they were transfused was established. This protocol allowed us to measure the effects of individual CCP transfusions on antibody levels in patients receiving CCP units with a wide range of antibody levels [16, 18, 19] . While usage of CCP has declined, 4 subjects in our study received CCP from vaccinated donors. This has allowed us to compare antibody responses with CCP from vaccinated and unvaccinated donors, providing data that could be used to design future studies related to the use of CCP for the treatment of COVID-19.

Methods and methods

Some COVID-19 patients were transfused under the expanded access protocol widely used in the United J o u r n a l P r e -p r o o f

Patient's admission and discharge date, age, sex, weight, and plasma unit volume were collected from electronic health records. The twenty five subjects in this study had an average age of 55.9 (median= 59) and ranged in age from 27 to 87. Nine of the 25 subjects were females (36%). Twenty-two of the patients received CCP within three days of hospitalization and three received the plasma after 2 days. Twelve subjects received 1 unit (including 4 who each received one unit of CCP from vaccinated donors) and 13

were transfused with 2 units. The mean number of symptomatic days prior to CCP transfusion was 10.7

(median=10.5) with a range of 2 to 23 days to treatment. All the patients in the study were discharged with the average days to discharge from CCP transfusion of 8.1 days (median=4) and range from 1 to 41 days. Patient charesteristics and a summary of the CCP dose they received is found in 

For this study, the antibody dose provided to each patient was calculated using the donor/unit antibody The patient's blood volume (L) was calculated from the patient's sex, height, and weight following the calculations routinely used for apheresis [21] . The plasma volume (L) was then calculated by multiplying the blood volume x (100% -Hematocrit). The predicted antibody level following transfusion was calculated by dividing the total CCP dose (kAU) by the patient's plasma volume (L) and adding this to the patient's SARS-CoV-2 antibody level (AU/ml) before transfusion.

Twenty-five CCP recipients have been tested for SARS-CoV-2 antibodies before (pre) and after (post) the CCP transfusion, and results for those receiving just one unit of CCP are shown in Figure 1A . Table 1 . In those receiving CCP from vaccinated donors, the average post transfusion antibody level was 210 AU/ml, a value higher even than the average value seen in CCP donors who had recovered from COVID-19 [16] . The results for those receiving 2 units of CCP are shown in Figure 1B . Nine of the 12 patients in this group had negative antibody levels before transfusion and 7 were antibody-positive after transfusion. Of the 7 antibody negative patients who received 2 units of CCP, the highest post transfusion antibody level detected was 61 AU/ml. All but one of the patients receiving 2 units of CCP showed an increase in antibody levels and the one exception was a patient whose antibody level prior to transfusion was 131 AU/ml. This value was higher than both of the CCP units they received so it is not surprising that their antibody level was reduced slightly by the transfusions. These results demonstrate that 1 unit of CCP from vaccinated donors with very high antibody levels provides a much larger antibody increment than even 2 units of CCP from unvaccinated donors.

We hypothesized that the amount of antibody in the CCP would correlate strongly with changes in antibody levels in CCP recipients. The antibody dose was calculated by multiplying the donor antibody level (AU/ml) by the unit volume (L). If two units were given, then these were simply added together. Figure 2 shows that the CCP dose strongly correlates with the difference in the patient post-CCP antibody level minus the pre-level (R 2 = 0.95). Of note, 7 of 7 seronegative patients receiving a dose greater than 50 kAU seroconverted after transfusion. In contrast, only 5 of 11 seronegative patients who received a CCP dose of less than 50 kAU seroconverted after transfusion.

We hypothesized that factoring in the patient's plasma volume, patient antibody level prior to transfusion, and the CCP dose may be used to predict the patient's antibody level after transfusion. For these calculations, the CCP dose (kAU) was divided by the patient's plasma volume (L) and this was then added to the patient's pre-transfusion antibody level (AU/ml). Figure 3 shows the predicted post-CCP vs actual post-CCP antibody levels for the patients in this study. This simple formula was surprisingly accurate in predicting these patient's antibody levels (R 2 = 0.92).

As a CCP program was established at our donor center, we suggested that CCP facilities use existing SARS-CoV-2 serological assays to screen CCP donors and collect samples from recipients for future research use [19] . Using this approach we found that the CCP dose (kAU) is a strong predictor of changes in antibody level in recipients of CCP. One primary determinant of the dose is the number of units received by the patient. Patients receiving just one unit of CCP from unvaccinated donors showed modest changes in antibody levels, whereas patients receiving 2 units of CCP from unvaccinated donors were much more likely to seroconvert. Focusing on recipients who were seronegative before transfusion, in those receiving a CCP dose of 50 kAU or higher, all 7 of these patients seroconverted, providing some insight into a threshold antibody titer that could be targeted when selecting units for transfusion. During the course of this study, the antibody level required for donation of CCP was increased from 30 to 100 AU/ml. With a value of 100 AU/ml and a minimum volume of 0.25 liters, the minimum dose of one unit of CCP would be 25 kAU (.25 x 100). Thus this data would indicate that using 2 units of CCP from donors with antibody levels > 100 AU/ml would be very likely to seroconvert recipients. While patientspecific results will certainly vary, this approach would provide a simple method of selecting CCP units with a high probability of leading to seroconversion following transfusion.

The FDA modified the CCP donor criteria to allow for collection of CCP from vaccinated donors on January 15, 2021. At this time we refocused our CCP collection efforts exclusively on vaccinated donors as we found these donors had exponentially higher antibody levels [16] . While nearly 20 vaccinated CCP donors have been tested to date, CCP was only collected from 6 due to donor restrictions outlined under the FDA guidance. Unfortunately, the donors who had the highest antibody responses (some in excess of 10,000 AU/ml) were more than 6 months out from recovery and therefore ineligible to donate CCP [16] .

The most striking findings in this report are the dramatic changes in antibody levels in the patients who received CCP from vaccinated donors. All had antibody levels following transfusion in excess of 100 AU/ml which is higher than the average levels seen in donors who had recovered from COVID-19 and far higher than the levels seen in the patients receiving CCP from unvaccinated donors. Whether these improved antibody responses with CCP from vaccinated donors result in improved outcomes is not known. This determination would require a well-controlled clinical trial focused on CCP from vaccinated donors. To this point, the data from the expanded access protocol which showed that patients who received "high-titer" CCP had improved survival would certainly support the hypothesis that vaccinated CCP could be superior to CCP from unvaccinated donors [15] . Of note, none of the CCP infused under the expanded access protocol could have come from vaccinated donors as CCP from vaccinated donors was not allowed until January of 2021, well after these patients had been treated.

To our knowledge, this is the first study that systematically examined antibody responses in patients receiving CCP and compared those results to the antibody units in the donors. While the optimal antibody level in COVID-19 patients is not known, a reasonable initial "transfusion goal" would be to seroconvert these patients following transfusion [18] . Our results show that a single unit of CCP from an unvaccinated donor commonly is not sufficient to achieve this result as 4 of 6 of these recipients remained seronegative after transfusion. Using 2 units of unvaccinted CCP or one unit of vaccinated CCP was much more successful in seroconverting recipients with 7 of 9 recipients seroconverting following 2 units of unvaccinated CCP and 3 of 3 recipients of vaccinated CCP seroconverting. Another possible "goal" for antibody levels following CCP treatment might be to achieve antibody levels commonly observed in patients recovered from COVID-19. In our studies, we found that CCP donors following recovery had an average spike specific antibody level of 82 AU/ml [16] . No seronegative patients receiving unvaccinated CCP had antibody levels this high. In contrast, the three seronegative patients who received CCP from vaccinated donors all had antibody levels over 100 AU/ml. Of note, one of these subjects had an estimated blood volume of over 8 L so these results could apply to nearly all patients independent of their size. These results demonstrate that a study using CCP from vaccinated donors could consistently achieve antibody levels commonly seen after recovery from COVID-19.

J o u r n a l P r e -p r o o f 

Convalescent plasma: possible therapy for novel coronavirus disease 2019

A consideration of convalescent plasma and plasma derivatives in the care of Severely-ill patients with COVID-19. Transfusion and apheresis science : official journal of the World Apheresis Association : official journal of the European Society for Haemapheresis

Elucidating the pivotal role of convalescent plasma therapy in critically ill COVID-19 patients: A review

Convalescent plasma in Covid-19: Possible mechanisms of action

Evaluation of current medical approaches for COVID-19: a systematic review and meta-analysis

A Scoping Review of Registered Clinical Trials of Convalescent Plasma for COVID-19 and a Framework for Accelerated Synthesis of Trial Evidence (FAST Evidence)

Clinical use of Convalescent Plasma in the COVID-19 pandemic; a transfusion-focussed gap analysis with recommendations for future research priorities

Infusion of Convalescent Plasma is associated with Clinical Improvement in Critically Ill Patients With Covid-19: A Comment

Effect of Convalescent Plasma Therapy on Time to Clinical Improvement in Patients With Severe and Life-threatening COVID-19: A Randomized Clinical Trial

Early High-Titer Plasma Therapy to Prevent Severe Covid-19 in Older Adults

A Randomized Trial of Convalescent Plasma in Covid-19 Severe Pneumonia

The Effect of Convalescent Plasma Therapy on Mortality Among Patients With COVID-19: Systematic Review and Meta-analysis

Mortality Benefit of Convalescent Plasma in COVID-19: A Systematic Review and Meta-Analysis

Efficacy and Safety of Convalescent Plasma for COVID-19: A Systematic Review and Meta-analysis

Convalescent Plasma Antibody Levels and the Risk of Death from Covid-19. The New England journal of medicine

Exponential increase in neutralizing and spike specific antibodies following vaccination of COVID-19 convalescent plasma donors

Antibody Responses after a Single Dose of SARS-CoV-2 mRNA Vaccine

COVID-19 Convalescent Plasma; Time for "Goal Directed Therapy"? Transfusion

COVID-19 convalescent plasma: phase 2

Head-to-Head Comparison of Two SARS-CoV-2 Serology Assays

Prediction of blood volume in normal human adults

We would like to thank Dena Voss who assisted in the collection and analysis of residual samples from