key: cord-321784-nubu5fuz
authors: Salazar, E.; Perez, K. K.; Ashraf, M.; Chen, J.; Castillo, B.; Christensen, P. A.; Eubank, T.; Bernard, D. W.; Eagar, T. N.; Long, S. W.; Subedi, S.; Olsen, R. J.; Leveque, C.; Schwartz, M. R.; Dey, M.; Chavez-East, C.; Rogers, J.; Shehabeldin, A.; Joseph, D.; Williams, G.; Thomas, K.; Masud, F.; Talley, C.; Dlouhy, K. G.; Lopez, B. V.; Hampton, C.; Lavinder, J.; Gollihar, J. D.; Maranhao, A. C.; Ippolito, G. C.; Saavedra, M. O.; Cantu, C. C.; Yerramilli, P.; Pruitt, L.; Musser, J. M.
title: Treatment of COVID-19 Patients with Convalescent Plasma in Houston, Texas
date: 2020-05-13
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2020.05.08.20095471
sha: 
doc_id: 321784
cord_uid: nubu5fuz

Background: COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for more than 100 years. Methods: Patients (n=25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28 to April 14, 2020. Patients were transfused with convalescent plasma obtained from donors with confirmed SARS-CoV-2 infection and had been symptom free for 14 days. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 post-transfusion. Clinical improvement was assessed based on a modified World Health Organization 6-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. Results: At baseline, all patients were receiving supportive care, including anti-inflammatory and anti-viral treatments, and all patients were on oxygen support. At day 7 post-transfusion with convalescent plasma, nine patients had at least a 1-point improvement in clinical scale, and seven of those were discharged. By day 14 post-transfusion, 19 (76%) patients had at least a 1-point improvement in clinical status and 11 were discharged. No adverse events as a result of plasma transfusion were observed. The whole genome sequencing data did not identify a strain genotype-disease severity correlation. Conclusions: The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease. Randomized, controlled trials are needed to determine its efficacy.

The coronavirus disease 2019 (COVID-19) pandemic has spread globally and caused massive loss of life and economic hardship. As of May 2, 2020, there were 3,494,671 confirmed cases and 246,475 deaths worldwide, and in the United States, there were 1,154,340 confirmed cases, and 67,447 deaths. 1 The disease is caused by SARS-CoV-2, a highly transmissible coronavirus first identified in Wuhan, China. [2] [3] [4] SARS-CoV-2 continues to spread in many countries, [5] [6] [7] [8] [9] and despite aggressive research, no proven therapies have been described.

Treatment strategies for critically ill COVID-19 patients are lacking with only limited evidence available for a battery of anti-viral, antibiotic, and anti-inflammatory agents and aggressive supportive therapy. Multiple clinical trials are ongoing, including the repurposing of remdesivir, an anti-viral investigated to treat Ebola, and hydroxychloroquine (HCQ), an antimalarial chloroquine derivative used to treat lupus and rheumatoid arthritis. There are early anti-COVID-19 efficacy data with remdesivir, 10 and preliminary data supporting the use of HCQ, alone or in combination with azithromycin (AZM), 11 has since been shown by larger controlled trials as misleading and potentially dangerous. 12 New therapies are needed to improve outcomes for critically ill COVID-19 patients.

In convalescent plasma therapy, blood plasma from a recovered patient is collected and transfused to a symptomatic patient. The transfer of convalescent plasma is an old concept, improvement in pulmonary lesions based on computed tomography (CT) scan. 19 A second study by Duan et al. reported improved clinical outcomes in 10 patients who received a single transfusion of convalescent plasma, and no adverse events reported. 18 Two additional small case studies of five and six patients have since been published with similar findings. 20,21 A more recent study by Zeng et. al. suggested that administration of convalescent plasma late in the disease course was ineffective for mortality reduction. 22 We performed the present study to provide additional data on these initial clinical observations of patients' clinical course and subsequent improvement after receiving convalescent plasma therapy for COVID-19. We transfused 25 COVID-19 patients with severe and/or life-threatening disease at the Houston Methodist hospitals, a large, quaternary-care hospital system that serves metropolitan Houston, Texas (~7 million people). 23 Patients were transfused once with 300 mL of convalescent plasma. The therapy was well-tolerated and no transfusion-related adverse events were observed. At day 7 post-transfusion, nine of 25 patients (36%) had improvement in the assessed clinical endpoints. By 14 days post-transfusion, 19 patients (76%) had improved or been discharged. Although our study has limitations, the data indicate that transfusion of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

CoV 28 and binds to the RBD of SARS-CoV-2 with a binding affinity of 6.3 nM. 29 Binding was performed at RT for 1 hr. Plates were washed and anti-human IgG Fab HRP (Sigma A0293, 1:5000) was added to the plate (50 µL), and incubated at RT for 30 min. Plates were washed 3X with PBST, ELISA substrate (1-step Ultra TMB, Thermo Scientific cat# 34028) was added, plates were developed for 1 min for RBD and 5 min for spike ECD, and the reaction was stopped with 50 µL of H2SO4. Plates were read at 450 nm absorbance. Three-fold serial dilutions from 50 to 4050 were analyzed. Titer was defined as the last dilution showing an optical density greater than a multi-plate negative control average plus six standard deviations.

Libraries for whole viral genome sequencing were prepared according to version 1 ARTIC nCoV-2019 sequencing protocol (https://artic.network/ncov-2019). Long reads were generated with the LSK-109 sequencing kit, 24 native barcodes (NBD104 and NBD114 kits), and a GridION instrument (Oxford Nanopore). Short reads were generated with the NexteraXT kit and a MiSeq or NextSeq 550 instrument (Illumina). Whole genome alignments of consensus viral genome sequence generated from the ARTIC nCoV-2019 bioinformatics pipeline were trimmed to the start of orf1ab and the end of orf10 and used to generate a phylogenetic tree using RAxML (https://cme.h-its.org/exelixis/web/software/raxml/index.html) to determine viral clade.

Trees were visualized and annotated with CLC Genomics Workbench v20 (Qiagen). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . https://doi.org/10.1101/2020.05.08.20095471 doi: medRxiv preprint hypertension (9 patients), hyperlipidemia (5 patients), and gastrointestinal reflux disease (GERD, 4 patients). The majority of patients (19 of 25, 76%) enrolled in the study had O-positive blood type. Bacterial or viral co-infections were identified in five patients (Table 1) .

The characteristics of the donors of convalescent plasma are shown in Table 2 . A total of nine donors provided plasma that was used to transfuse COVID-19 patients; two donors gave plasma on multiple occasions. The donors ranged in age from 23 to 67 years, and 56% (5/9) were males. On average, the donors gave plasma 26 days (range 19-33) from their symptom start date and 21 days (range 13 to 27 days) from their initial positive RT-PCR specimen collection date. Although all donors had been symptomatic, only one was ill enough to require hospitalization. To assess antibody titers, we used two ELISAs, one based on recombinant purified ectodomain (ECD) of the spike protein and the second using recombinant receptor binding domain (RBD) of the spike protein. The titers of the convalescent plasma used for transfusion ranged from 0 to 1350 for the RBD and ECD domains (Figure 1 and Table S1 ).

The median time from symptom onset to hospitalization was 6 days [IQR 4 to 8 days] ( Table 3) .

The majority of patients received concomitant anti-inflammatory treatments within five days of the plasma transfusion, including tocilizumab and steroids. Most received other investigational treatments, including courses of HCQ and AZM, ribavirin, and/or lopinavir/ritonavir, and two patients received remdesivir ( Table 3) . All patients required oxygen support prior to transfusion (Figure 1) , including 12 patients on mechanical ventilation, 10 on low-flow oxygen, and 3 on high-flow oxygen. One patient (patient 9) was placed on ECMO on the day of transfusion prior to transfusion. More than half (13/25, 52%) had acute respiratory distress syndrome (ARDS) 30 at the time of transfusion ( Table 3 ). The median time from symptom onset to transfusion was 10 days [IQR, 7.5 to 12.5], and from hospitalization to transfusion was 2 days [IQR, 2 to 4] (Table All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . https://doi.org/10.1101/2020.05.08.20095471 doi: medRxiv preprint There was a trend toward increasing ferritin by day 3, which tended to decrease by day 7. No significant increases in liver enzymes were noted ( Table 4 and Table S3 ).

A recent analysis of the genomic heterogeneity of the SARS-CoV-2 virus strains circulating in Houston, Texas, early in the pandemic showed that the predominant clades isolated were A2a, B, and B1. 32 Amino acid polymorphisms, especially in the spike protein, can potentially alter the character of the antibody response and virulence profile of the virus. 33-36 Therefore, we sequenced the genomes of the SARS-CoV-2 virus strains infecting donors and recipients to assess the magnitude of nucleotide and amino acid mismatch between the viral genotype of donors and plasma recipients. Of the 34 patients and donors, we were able to analyze all plasma recipient genotypes and four donor genotypes. Overall, there were few polymorphisms in the sequenced viruses, and there was no correlation between infecting strains and disease severity ( Figure S1 ). Analysis of the first four donors found that, in general, donor and recipient S proteins matched when their SARS-CoV2 isolates were from the same clade ( Figure S1 ). This is primarily a result of a D614G amino acid change in S protein that defines the clade A2a. 32,37 However, there are at least three instances of an additional amino acid change in the S2 domain of the S protein, 34-36 one in a donor (M731I) and two in recipients (S967R and L1203F) ( Figure S1 ).

Our study was performed to evaluate the safety and potential benefit of transfusing convalescent plasma to patients with severe COVID-19 disease. To date, this is the largest cohort assessed for outcomes pertaining to convalescent plasma transfusion for COVID-19. Of our 25 patients, nine showed improvement by day 7, and by day 14 post-transfusion, 19

patients showed improvement, as assessed by discharge or at least a 1-point improvement on a modified clinical scale. Several case studies investigating the use of convalescent plasma to All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . https://doi.org/10.1101/2020.05.08.20095471 doi: medRxiv preprint treat severe COVID-19 have recently been published 18-22 and the overall findings presented herein are consistent with these reports.

Convalescent plasma therapy has been administered on the front lines during emergencies, and we and others recognize the need for controlled clinical trials to determine its therapeutic efficacy. 14, 15, 19, 38, 39 The timing of the transfusion post-symptom onset, the number of transfusions, the volume and its adjustment based on BMI, donor antibody titers, and other parameters need to be evaluated to optimize this therapy. For example, some studies have observed that the sooner after the onset of symptoms that the transfusion was administered, the better the outcomes. 14, 15, 39 Variability existed among our cohort with respect to symptom onset and severity of illness.

The anti-SARS-CoV2 anti-spike protein IgG titers varied significantly among individual donors, as assessed by ELISA (Table S1 ). Early in the study period, ELISA titers were not available, and thus, transfusions were given solely on the basis of ABO compatibility. Among the five patients who received plasma from a donor with an anti-RBD IgG titer of ≤50, one is The results from our study support the existing data from the COVID-19 literature that point to underlying medical conditions, such as obesity, type 2 diabetes, and hypertension, playing a large role in patients' COVID-19 disease course and outcomes. [40] [41] [42] Sixty-eight percent (17/25) of transfused patients in this study had a BMI in the obese category and 84% were considered overweight. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . https://doi.org/10.1101/2020.05.08.20095471 doi: medRxiv preprint A confounding variable in many convalescent plasma studies is the addition of other treatment regimens, such as antivirals and anti-inflammatory compounds. Adjunct therapies hinder the ability to draw definitive conclusions regarding the contribution of the convalescent plasma. In our study, all 25 patients received HCQ and AZM, as these were reported to have beneficial effects early in the pandemic. 11 Subsequent larger and more controlled studies determined that this combination has no benefits to patients, and in fact, could be harmful. 12

Many (68%) of our patients were also administered oral ribavirin. Despite inconclusive data on ribavirin's efficacy in the treatment of SARS during the 2003 epidemic, 43 proven safety and ready availability supported its use in the treatment of our COVID-19 patients. Two patients received remdesivir, which was recently shown to modestly reduce recovery time. 10,44 Antiinflammatory compounds, such as the IL-6 inhibitor tocilizumab and methylprednisolone, were administered per institutional protocols within five days of the plasma transfusion to 72% of our cohort. Tocilizumab was recently shown to reduce mortality in a retrospective analysis of 20 severe COVID-19 patients. 45 Because convalescent plasma therapy is typically performed in emergency situations for the very ill, it is difficult to assess its benefits as a standalone treatment. A blinded, randomized controlled trial is currently being considered.

The patient outcomes in our study are similar to those recently published describing treating COVID-19 patients with remdesivir on a compassionate-use basis. 10 In that review, patients were prescribed a 10-day course of remdesivir with follow up for 28 days or until discharge or death. Both study cohorts included patients who required invasive ventilation, (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . https://doi.org/10.1101/2020.05.08.20095471 doi: medRxiv preprint mortality difference between the cohorts cannot be compared as the remdesivir cohort represented an older population (median age of 64 years, versus 51 years in our study), where the risk of death was greater at baseline. Delays in obtaining remdesivir on a compassionate use basis (12 days from symptom onset) may have artificially extended the cohort's opportunity to demonstrate clinical improvement and does not reflect the eligibility criteria for any ongoing clinical trials. [46] [47] [48] Clinical outcomes data to inform timing of therapeutic interventions like remdesivir or convalescent plasma are lacking.

We analyzed the genomes of the infecting SARS-CoV-2 strain from both the donors and recipients. One could conceive of a situation in which the donor genotype of the SARS-CoV-2 infecting strain was matched with the genotype of the patient's strain to maximize potential immune benefit. We found few differences in the inferred amino acid sequences of the plasma donor and recipient strains, and no association between disease severity and infecting strain genotype.

The majority of the donors and plasma recipients in our study had type O blood (25/34, 74%). Our initial donors, who donated repeatedly, were blood type O. Since ABO-compatibility was a requirement for recipient selection early in the study, many of our early recipients were also type O. Zhao et al. have reported that of the 2,173 patients analyzed in their study of COVID-19 patients in China, the majority had type A blood. 49 More studies are needed to determine if this association holds true in geographically-distinct areas of infection. Regardless, our data do not reflect a higher rate of blood type A in COVID-19 patients.

As with the great majority of the studies using convalescent plasma to treat severe infections, our study has several important limitations. First, the study was a small case series and no control group was included. Thus, it is not clear if the 25 patients given convalescent plasma would have improved without this treatment. Second, all patients were treated with multiple other medications, including antiviral and anti-inflammatory agents. Thus, we cannot conclude All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . https://doi.org/10.1101/2020.05.08.20095471 doi: medRxiv preprint that the patient outcomes were solely due to administration of convalescent plasma. Third, 24 of the 25 patients received only one transfusion of plasma. Whether treatment with multiple transfusions on one or more days would be a more effective regimen is not clear. An expanded donor pool providing higher-titer convalescent plasma would allow for dose escalation studies.

Fourth, many patients had severe COVID-19 disease. It is possible that transfusion of convalescent plasma earlier in the course of disease or in patients with less severe symptoms would be a better approach. Fifth, our plasma donors had a range of anti-S protein IgG titers.

Several patients were transfused with plasma with very low titer of anti-S protein antibody. Sixth, the small number of patients treated, coupled with the experimental design, did not permit us to determine if this therapy significantly reduces mortality or other measures of disease outcome.

Finally, while this study assessed outcomes at days 7 and 14 post-transfusion, it is important to note that at the time of this writing, all but two of the surviving patients that were intubated had been extubated. Similarly, all patients that were on ECMO had been weaned, and 20 of the 25 patients had been discharged.

Outcomes from this case series of 25 patients indicates that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . https://doi.org/10.1101/2020.05.08.20095471 doi: medRxiv preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 13, 2020. . https://doi.org/10.1101/2020.05.08.20095471 doi: medRxiv preprint

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