In vivo genotoxicity assessment of N-(-9 acridinyl)-b-alanine hydrochloride (S-300) using a validated Pig-a mutagenesis assay.

BACKGROUND: N-(-9 acridinyl)-b-alanine hydrochloride (S-300) is the main byproduct of red blood cell (RBC) amustaline/glutathione(GSH) pathogen reduction, currently undergoing phase III US clinical trials following successful European studies(1-3). Phosphatidylinositol glycan, class A (Pig-a) X-linked gene mutagenesis is a validated mammalian in vivo mutation assay for genotoxicity, assessed as clonal loss of glycosylphosphatidylinositol-linked CD59 cell-surface molecules on reticulocytes (RETs) and RBCs. METHODS: Male Sprague-Dawley rats received continuous infusion of S-300 up to the maximum feasible dose (240 mg/kg/day-limited by solubility and volume) for 28 days. Positive controls received a known mutagen by oral gavage on Days 1-3. Plasma levels of S-300 were assessed by HPLC before, during and after infusion. CD59-negative RBCs and RETs were enumerated in pre-dose and Day 28 samples, using a flow cytometric method. Outcome was evaluated by predetermined criteria using concurrent and historical controls. Toxicity was assessed by laboratory measures and necropsy. RESULTS: S-300 reached maximum, dose-dependent levels (3-15 µmol/L) within 2-8 h that were sustained for 672 h and undetectable 2 h after infusion. Circulating RET levels indicated a lack of hematopoietic toxicity. Necropsy revealed minimal-mild observations related to poor S-300 solubility at high concentrations. Pig-a assessment met the preset acceptability criteria and revealed no increase in mutant RBCs or RETs. CONCLUSIONS: Maximum feasible S-300 exposure of rats by continuous infusion for 28 days was not genotoxic as assessed by an Organization for Economic Cooperation and Development-compliant, mammalian, in vivo Pig-a gene mutation assay that meets the requirements of International Conference on Harmonization (ICH) S2(R1) and FDA guidances on genotoxicity testing.

Acute pulmonary injury in hematology patients supported with pathogen-reduced and conventional platelet components.

ABSTRACT: Patients treated with antineoplastic therapy often develop thrombocytopenia requiring platelet transfusion, which has potential to exacerbate pulmonary injury. This study tested the hypothesis that amotosalen-UVA pathogen-reduced platelet components (PRPCs) do not potentiate pulmonary dysfunction compared with conventional platelet components (CPCs). A prospective, multicenter, open-label, sequential cohort study evaluated the incidence of treatment-emergent assisted mechanical ventilation initiated for pulmonary dysfunction (TEAMV-PD). The first cohort received CPC. After the CPC cohort, each site enrolled a second cohort transfused with PRPC. Other outcomes included clinically significant pulmonary adverse events (CSPAE) and the incidence of treatment-emergent acute respiratory distress syndrome (TEARDS) diagnosed by blinded expert adjudication. The incidence of TEAMV-PD in all patients (1068 PRPC and 1223 CPC) was less for PRPC (1.7 %) than CPC (3.1%) with a treatment difference of -1.5% (95% confidence interval [CI], -2.7 to -0.2). In patients requiring ≥2 PCs, the incidence of TEAMV-PD was reduced for PRPC recipients compared with CPC recipients (treatment difference, -2.4%; 95% CI, -4.2 to -0.6). CSPAE increased with increasing PC exposure but were not significantly different between the cohorts. For patients receiving ≥2 platelet transfusions, TEARDS occurred in 1.3% PRPC and 2.6% CPC recipients (P = .086). Bayesian analysis demonstrated PRPC may be superior in reducing TEAMV-PD and TEARDS for platelet transfusion recipients compared with CPC recipients, with 99.2% and 88.8% probability, respectively. In this study, PRPC compared with CPC demonstrated high probability of reduced severe pulmonary injury requiring assisted mechanical ventilation in patients with hematology disorders dependent on platelet transfusion. This trial was registered at www.ClinicalTrials.gov as #NCT02549222.

Evaluation of the efficacy and safety of amustaline/glutathione pathogen-reduced RBCs in complex cardiac surgery: the Red Cell Pathogen Inactivation (ReCePI) study-protocol for a phase 3, randomized, controlled trial.

BACKGROUND: Red blood cell (RBC) transfusion is a critical supportive therapy in cardiovascular surgery (CVS). Donor selection and testing have reduced the risk of transfusion-transmitted infections; however, risks remain from bacteria, emerging viruses, pathogens for which testing is not performed and from residual donor leukocytes. Amustaline (S-303)/glutathione (GSH) treatment pathogen reduction technology is designed to inactivate a broad spectrum of infectious agents and leukocytes in RBC concentrates. The ReCePI study is a Phase 3 clinical trial designed to evaluate the efficacy and safety of pathogen-reduced RBCs transfused for acute anemia in CVS compared to conventional RBCs, and to assess the clinical significance of treatment-emergent RBC antibodies. METHODS: ReCePI is a prospective, multicenter, randomized, double-blinded, active-controlled, parallel-design, non-inferiority study. Eligible subjects will be randomized up to 7 days before surgery to receive either leukoreduced Test (pathogen reduced) or Control (conventional) RBCs from surgery up to day 7 post-surgery. The primary efficacy endpoint is the proportion of patients transfused with at least one study transfusion with an acute kidney injury (AKI) diagnosis defined as any increased serum creatinine (sCr) level ≥ 0.3 mg/dL (or 26.5 µmol/L) from pre-surgery baseline within 48 ± 4 h of the end of surgery. The primary safety endpoints are the proportion of patients with any treatment-emergent adverse events (TEAEs) related to study RBC transfusion through 28 days, and the proportion of patients with treatment-emergent antibodies with confirmed specificity to pathogen-reduced RBCs through 75 days after the last study transfusion. With ≥ 292 evaluable, transfused patients (> 146 per arm), the study has 80% power to demonstrate non-inferiority, defined as a Test group AKI incidence increase of no more than 50% of the Control group rate, assuming a Control incidence of 30%. DISCUSSION: RBCs are transfused to prevent tissue hypoxia caused by surgery-induced bleeding and anemia. AKI is a sensitive indicator of renal hypoxia and a novel endpoint for assessing RBC efficacy. The ReCePI study is intended to demonstrate the non-inferiority of pathogen-reduced RBCs to conventional RBCs in the support of renal tissue oxygenation due to acute anemia and to characterize the incidence of treatment-related antibodies to RBCs.

Leveraging Donor Populations to Study the Epidemiology and Pathogenesis of Transfusion-Transmitted and Emerging Infectious Diseases.

The tragedy of transfusion-associated hepatitis and HIV spurred a decades-long overhaul of the regulatory oversight and practice of blood transfusion. Consequent to improved donor selection, testing, process control, clinical transfusion practice and post-transfusion surveillance, transfusion in the United States and other high-income countries is now a very safe medical procedure. Nonetheless, pathogens continue to emerge and threaten the blood supply, highlighting the need for a proactive approach to blood transfusion safety. Blood donor populations and the global transfusion infrastructure are under-utilized resources for the study of infectious diseases. Blood donors are large, demographically diverse subsets of general populations for whom cross-sectional and longitudinal samples are readily accessible for serological and molecular testing. Blood donor collection networks span diverse geographies, including in low- and middle-income countries, where agents, especially zoonotic pathogens, are able to emerge and spread, given limited tools for recognition, surveillance and control. Routine laboratory storage and transportation, coupled with data capture, afford access to rich epidemiological data to assess the epidemiology and pathogenesis of established and emerging infections. Subsequent to the State of the Science in Transfusion Medicine symposium in 2022, our working group (WG), "Emerging Infections: Impact on Blood Science, the Blood Supply, Blood Safety, and Public Health" elected to focus on "leveraging donor populations to study the epidemiology and pathogenesis of transfusion-transmitted and emerging infectious diseases." The 5 landmark studies span (1) the implication of hepatitis C virus in post-transfusion hepatitis, (2) longitudinal evaluation of plasma donors with incident infections, thus informing the development of a widely used staging system for acute HIV infection, (3) explication of the dynamics of early West Nile Virus infection, (4) the deployment of combined molecular and serological donor screening for Babesia microti, to characterize its epidemiology and infectivity and facilitate routine donor screening, and (5) national serosurveillance for SARS-CoV-2 during the COVID-19 pandemic. The studies highlight the interplay between infectious diseases and transfusion medicine, including the imperative to ensure blood transfusion safety and the broader application of blood donor populations to the study of infectious diseases.

Characterization of antibodies to SARS-CoV-2 in lyophilized plasma prepared with amotosalen-UVA pathogen reduction.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-infected patients exhibit disease ranging from asymptomatic to severe pneumonia, multi-organ failure, and death. convalescent COVID plasma (CCP) from recovered patients with high levels of neutralizing antibodies has demonstrated therapeutic efficacy to reduce the morbidity of coronavirus disease 2019 (COVID-19) in some studies. The development of assays to characterize the activity of CCP to neutralize SARS-CoV-2 infectivity offers the possibility to improve potential therapeutic efficacy. Lyophilization of CCP may increase the availability of this therapy. We hypothesized that SARS-CoV-2 antibody profiles of pooled lyophilized pathogen-reduced CCP from COVID-19-recovered blood donors retains virus-neutralizing efficacy as reported for frozen pathogen-reduced CCP. METHODS: Pooled lyophilized pathogen-reduced plasma was prepared from recovered COVID plasma donors. Antibodies to SARS-CoV-2 were characterized in each donor plasma prior to pathogen reduction and lyophilization and after lyophilization of individual CCP, and in the lyophilized CCP pool. Several complimentary assays were used to characterize antibody levels, neutralizing capacity, and the spectrum of antigen reactivity. The mean values for individual plasma samples and the value in the pool were compared. RESULTS: The mean ratio for antibody binding to SARS-CoV-2 antigens before and after treatment was 0.95 ± 0.22 mean fluorescent intensity (MFI) units. Antibody activity to an array of influenza virus antigens demonstrated a mean activity ratio of 0.92 ± 0.12 MFI before and after treatment. CONCLUSIONS: The antibody activity in pooled pathogen-reduced lyophilized CCPs demonstrated minimal impact due to pathogen reduction treatment and lyophilization.

Immediate use cryoprecipitate products provide lasting organ protection in a rodent model of trauma/hemorrhagic shock and prolonged hypotensive resuscitation.

BACKGROUND: Cryoprecipitate (CP) can augment hemostasis after hemorrhagic shock (HS). Similar to fresh frozen plasma (FFP), CP may provide short-term endothelial protection. We tested a new 5-day postthaw CP (5-day pathogen-reduced cryoprecipitate [5PRC]) and lyophilized pathogen-reduced cryoprecipitate (LPRC) to overcome challenges of early administration and hypothesized that 5PRC and LPRC would provide lasting organ protection in a rodent model of HS. METHODS: Mice underwent trauma/HS (laparotomy then HS), mean arterial pressure (MAP) 35 × 90 minutes, and then 6 hours of hypotensive resuscitation (MAP, 55-60 mm Hg) with lactated Ringer's solution (LR), FFP, CP, 5PRC, or LPRC and compared with shams. Animals were followed for 72 hours. Organs and blood were collected. Data are presented as mean ± SD and analysis of variance with Bonferroni post hoc. RESULTS: Mean arterial pressure was comparable between experimental groups at baseline, preresuscitation, and 6 hours per protocol. However, volume needed to resuscitate to target MAP over 6 hours was less than half for CP, 5PRC, LPRC, and FFP compared with LR, suggesting that CP products can serve as effective resuscitative agents. Mean arterial pressure at 72 hours was also significantly higher in the CP, 5PRC, and FFP groups compared with LR. Resuscitation with CP, 5PRC, and LPRC provided lasting protection from gut injury and enhanced syndecan immunostaining comparable with FFP, while LR mice demonstrated persistent organ dysfunction. Sustained endothelial protection was demonstrated by lessened lung permeability, while cystatin C was an indicator of kidney function, and liver aspartate aminotransferase and alanine transaminase returned to sham levels in all groups. CONCLUSION: Cryoprecipitate products can provide lasting organ protection comparable with FFP in a sustained rodent model of trauma/HS and hypotensive resuscitation. The availability of 5PRC and LPRC will allow for investigation into the immediate use of cryoprecipitate for severely injured patients. As lyophilized products such as cryoprecipitate become available clinically, their use has important implications for prehospital, rural, and battlefield usage.

Early lyophilized cryoprecipitate enhances the ADAMTS13/VWF ratio to reduce systemic endotheliopathy and lessen lung injury in a mouse multiple-trauma hemorrhage model.

BACKGROUND: Recent studies in severely injured patients suggest an important role of von Willebrand Factor (VWF) and ADAMTS13 in the endotheliopathy of trauma (EoT). We hypothesized that the early use of cryoprecipitate would be effective as an endothelial protector by supplementing physiologic VWF and ADAMTS13 to reverse the EoT. We tested a pathogen-reduced lyophilized cryoprecipitate (LPRC) that could expedite the early administration of cryoprecipitate in the battlefield. METHODS: A mouse multiple-trauma model with uncontrolled hemorrhage (UCH) from liver injury was utilized followed by hypotensive resuscitation (mean arterial pressure, 55-60) × 3 hours with lactated Ringer's (LR), fresh frozen plasma (FFP), conventional pathogen-reduced cryoprecipitate (CC), and LPRC. Blood was collected for measurement of syndecan-1, VWF, and ADAMTS13 by ELISA. Lungs were stained for histopathologic injury and syndecan-1 and bronchial alveolar lavage (BAL) fluid harvested for protein as an indicator of permeability. Statistical analysis was by ANOVA followed by Bonferroni correction. RESULTS: Following multiple trauma and UCH, blood loss was similar across groups. Mean volume of resuscitation was higher in the LR group compared with the other resuscitation groups. Lung histopathologic injury, syndecan-1 immunostaining and BAL protein were higher with LR compared with resuscitation with FFP and CC, while LPRC further reduced BAL compared with FFP and CC. The ADAMTS13/VWF ratio was significantly lower in LR but improved with FFP and CC, comparable to shams while LPRC further increased this ratio. CONCLUSION: The protective effects of CC and LPRC were comparable to FFP in ameliorating the EoT in our murine multiple trauma and UCH model. Lyophilized cryoprecipitate may also provide additional benefit by enhancing the ADAMTS13/VWF ratio. These data provide evidence of the safety and efficacy of LPRC and warrants further investigation for its potential application in military settings once approved for human administration.

Longitudinal analysis of annual national hemovigilance data to assess pathogen reduced platelet transfusion trends during conversion to routine universal clinical use and 7-day storage.

BACKGROUND: France converted to universal pathogen reduced (PR; amotosalen/UVA) platelets in 2017 and extended platelet component (PC) shelf-life from 5- to 7-days in 2018 and 2019. Annual national hemovigilance (HV) reports characterized longitudinal PC utilization and safety over 11 years, including several years prior to PR adoption as the national standard of care. METHODS: Data were extracted from published annual HV reports. Apheresis and pooled buffy coat [BC] PC use was compared. Transfusion reactions (TRs) were stratified by type, severity, and causality. Trends were assessed for three periods: Baseline (2010-14; ~7% PR), Period 1 ([P1] 2015-17; 8%-21% PR), and Period 2 ([P2] 2018-20; 100% PR). RESULTS: PC use increased by 19.1% between 2010 and 2020. Pooled BC PC production increased from 38.8% to 68.2% of total PCs. Annual changes in PCs issued averaged 2.4% per year at baseline, -0.02% (P1) and 2.8% (P2). The increase in P2 coincided with a reduction in the target platelet dose and extension to 7-day storage. Allergic reactions, alloimmunization, febrile non-hemolytic TRs, immunologic incompatibility, and ineffective transfusions accounted for >90% of TRs. Overall, TR incidence per 100,000 PCs issued declined from 527.9 (2010) to 345.7 (2020). Severe TR rates declined 34.8% between P1-P2. Forty-six transfusion-transmitted bacterial infections (TTBI) were associated with conventional PCs during baseline and P1. No TTBI were associated with amotosalen/UVA PCs. Infections with Hepatitis E (HEV) a non-enveloped virus resistant to PR, were reported in all periods. DISCUSSION: Longitudinal HV analysis demonstrated stable PC utilization trends with reduced patient risk during conversion to universal 7-day amotosalen/UVA PCs.

Characterization of pathogen-inactivated COVID-19 convalescent plasma and responses in transfused patients.

BACKGROUND: Efficacy of donated COVID-19 convalescent plasma (dCCP) is uncertain and may depend on antibody titers, neutralizing capacity, timing of administration, and patient characteristics. STUDY DESIGN AND METHODS: In a single-center hypothesis-generating prospective case-control study with 1:2 matched dCCP recipients to controls according to disease severity at day 1, hospitalized adults with COVID-19 pneumonia received 2 × 200 ml pathogen-reduced treated dCCP from 2 different donors. We evaluated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in COVID-19 convalescent plasma donors and recipients using multiple antibody assays including a Coronavirus antigen microarray (COVAM), and binding and neutralizing antibody assays. Outcomes were dCCP characteristics, antibody responses, 28-day mortality, and dCCP -related adverse events in recipients. RESULTS: Eleven of 13 dCCPs (85%) contained neutralizing antibodies (nAb). PRT did not affect dCCP antibody activity. Fifteen CCP recipients and 30 controls (median age 64 and 65 years, respectively) were enrolled. dCCP recipients received 2 dCCPs from 2 different donors after a median of one hospital day and 11 days after symptom onset. One dCCP recipient (6.7%) and 6 controls (20%) died (p = 0.233). We observed no dCCP-related adverse events. Transfusion of unselected dCCP led to heterogeneous SARS CoV-2 antibody responses. COVAM clustered dCCPs in 4 distinct groups and showed endogenous immune responses to SARS-CoV-2 antigens over 14-21 days post dCCP in all except 4 immunosuppressed recipients. DISCUSSION: PRT did not impact dCCP anti-virus neutralizing activity. Transfusion of unselected dCCP did not impact survival and had no adverse effects. Variable dCCP antibodies and post-transfusion antibody responses indicate the need for controlled trials using well-characterized dCCP with informative assays.

Evaluation of amotosalen and UVA pathogen-reduced apheresis platelets after 7-day storage.

BACKGROUND: Amotosalen/UVA pathogen-reduced platelet components (PRPCs) with storage up to 7 days are standard of care in France, Switzerland, and Austria. PRPCs provide effective hemostasis with reduced risk of transfusion-transmitted infections and transfusion-associated graft versus host disease, reduced wastage and improved availability compared with 5-day-stored PCs. This study evaluated the potency of 7-day PRPCs by in vitro characterization and in vivo pharmacokinetic analysis of autologous PCs. STUDY DESIGN AND METHODS: The in vitro characteristics of 7-day-stored apheresis PRPCs suspended in 100% plasma or 65% platelet additive solution (PAS-3)/35% plasma, thrombin generation, and in vivo radiolabeled post-transfusion recovery and survival of 7-day-stored PRPCs suspended in 100% plasma were compared with either 7-day-stored or fresh autologous conventional platelets. RESULTS: PRPCs after 7 days of storage maintained pH, platelet dose, in vitro physiologic characteristics, and thrombin generation when compared to conventional 7-day PCs. In vivo, the mean post-transfusion survival was 151.4 ± 20.1 h for 7-day PRPCs in 100% plasma (Test) versus 209.6 ± 13.9 h for the fresh autologous platelets (Control), (T-ΔC: 72.3 ± 8.8%: 95% confidence interval [CI]: 68.5, 76.1) and mean 24-h post-transfusion recovery 37.6 ± 8.4% for Test versus 56.8 ± 9.2% for Control (T-ΔC: 66.2 ± 11.2%; 95% CI: 61.3, 71.1). DISCUSSION: PRPCs collected in both 100% plasma as well as 65% PAS-3/35% plasma and stored for 7 days retained in vitro physiologic characteristics. PRPCs stored in 100% plasma for 7 days retained in vivo survival. Lower in vivo post-radiolabeled autologous platelet recovery is consistent with reported reduced count increments for allogenic transfusion.

Detection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR.

An easily implementable serological assay to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies is urgently needed to better track herd immunity, vaccine efficacy and vaccination rates. Herein, we report the Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) which uses real-time qPCR to measure the ability of neutralizing antibodies to block binding between DNA-barcoded viral spike protein subunit 1 and the human angiotensin-converting enzyme 2 receptor protein. The SONIA neutralizing antibody assay using finger-prick dried blood spots displays 91-97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern.

Comparative risk of pulmonary adverse events with transfusion of pathogen reduced and conventional platelet components.

BACKGROUND: Platelet transfusion carries risk of transfusion-transmitted infection (TTI). Pathogen reduction of platelet components (PRPC) is designed to reduce TTI. Pulmonary adverse events (AEs), including transfusion-related acute lung injury and acute respiratory distress syndrome (ARDS) occur with platelet transfusion. STUDY DESIGN: An open label, sequential cohort study of transfusion-dependent hematology-oncology patients was conducted to compare pulmonary safety of PRPC with conventional PC (CPC). The primary outcome was the incidence of treatment-emergent assisted mechanical ventilation (TEAMV) by non-inferiority. Secondary outcomes included: time to TEAMV, ARDS, pulmonary AEs, peri-transfusion AE, hemorrhagic AE, transfusion reactions (TRs), PC and red blood cell (RBC) use, and mortality. RESULTS: By modified intent-to-treat (mITT), 1068 patients received 5277 PRPC and 1223 patients received 5487 CPC. The cohorts had similar demographics, primary disease, and primary therapy. PRPC were non-inferior to CPC for TEAMV (treatment difference -1.7%, 95% CI: (-3.3% to -0.1%); odds ratio = 0.53, 95% CI: (0.30, 0.94). The cumulative incidence of TEAMV for PRPC (2.9%) was significantly less than CPC (4.6%, p = .039). The incidence of ARDS was less, but not significantly different, for PRPC (1.0% vs. 1.8%, p = .151; odds ratio = 0.57, 95% CI: (0.27, 1.18). AE, pulmonary AE, and mortality were not different between cohorts. TRs were similar for PRPC and CPC (8.3% vs. 9.7%, p = .256); and allergic TR were significantly less with PRPC (p = .006). PC and RBC use were not increased with PRPC. DISCUSSION: PRPC demonstrated reduced TEAMV with no excess treatment-related pulmonary morbidity.

Is pathogen reduction an acceptable alternative to irradiation for risk mitigation of transfusion-associated graft versus host disease?

Transfusion-associated graft versus host disease (TA-GVHD) is a highly morbid and often fatal adverse event associated with transfusion of cellular blood products [platelets, red blood cells (RBCs) and whole blood] and more rarely with never-frozen plasma products. It is caused by residual viable donor T-lymphocytes that proliferate and actively target recipient tissues. Selective or universal irradiation of blood components using gamma-irradiation and more recently, X-ray irradiation, are the most commonly applied interventions and have been validated by the demonstration of in vitro T-lymphocyte inactivation, in murine models of TA-GVHD and by years of clinical experience. Irradiation, however, has multiple limitations including a sharp dose-response curve that renders quality control of dosage critically important, the use of radioactive radiation sources that are a terrorism risk, and selective implementation in many countries that leads to inadvertent omission and patient risk exposure. Certain pathogen reduction technologies (PRT) for platelets have been approved by regulatory authorities and endorsed by professional societies as an alternative to irradiation for reducing the risk of TA-GVHD, and PRT for RBCs and whole blood are in development. While the mechanism of action of T-lymphocyte inactivation differs from gamma/X-ray irradiation, the impact on T-lymphocyte inactivation for PRT is equivalent or superior to that of irradiation as demonstrated by sensitive in vitro lymphocyte proliferation assays and in vivo mouse models that approximate human TA-GVHD. Clinical trials and cumulative routine-use experience attest to the efficacy of PRT when used as an alternative to irradiation. While T-lymphocyte inactivation efficacy varies between PRT platforms, the implementation of PRT for platelets increases blood safety for patients beyond the mitigation of TA-GVHD, by decreasing the risk of transfusion transmitted infections with known viruses, bacteria and parasites as well as emerging pathogens.

Antibody profiles in COVID-19 convalescent plasma prepared with amotosalen/UVA pathogen reduction treatment.

BACKGROUND: COVID-19 convalescent plasma (CCP), from donors recovered from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, is one of the limited therapeutic options currently available for the treatment of critically ill patients with COVID-19. There is growing evidence that CCP may reduce viral loads and disease severity; and reduce mortality. However, concerns about the risk of transfusion-transmitted infections (TTI) and other complications associated with transfusion of plasma, remain. Amotosalen/UVA pathogen reduction treatment (A/UVA-PRT) of plasma offers a mitigation of TTI risk, and when combined with pooling has the potential to increase the diversity of the polyclonal SARS-CoV-2 neutralizing antibodies. STUDY DESIGN AND METHODS: This study assessed the impact of A/UVA-PRT on SARS-CoV-2 antibodies in 42 CCP using multiple complimentary assays including antigen binding, neutralizing, and epitope microarrays. Other mediators of CCP efficacy were also assessed. RESULTS: A/UVA-PRT did not negatively impact antibodies to SARS-CoV-2 and other viral epitopes, had no impact on neutralizing activity or other potential mediators of CCP efficacy. Finally, immune cross-reactivity with other coronavirus antigens was observed raising the potential for neutralizing activity against other emergent coronaviruses. CONCLUSION: The findings of this study support the selection of effective CCP combined with the use of A/UVA-PRT in the production of CCP for patients with COVID-19.

Distinct SARS-CoV-2 antibody reactivity patterns in coronavirus convalescent plasma revealed by a coronavirus antigen microarray.

A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and random forest (RF) prediction model, to classify individual specimens as either Reactive or non-reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the three assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 91%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed three main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified by COVAM reactivity patterns offers potential to improve CCP therapeutic efficacy CoV2T alone. The use of a SARS-CoV-2 antigen array can qualify CCP for administration as a treatment for acute COVID-19, and interrogate vaccine immunogenicity and performance in preclinical, clinical studies, and routine vaccination to identify antibody responses predictive of protection from infection and disease.

Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Prevalence of natural and acquired antibodies to amustaline/glutathione pathogen reduced red blood cells.

BACKGROUND: The INTERCEPT™ Blood System for Red Blood Cells (RBCs) utilizes amustaline (S-303) and glutathione (GSH) to inactivate pathogens and leukocytes in transfused RBCs. Treatment-emergent low titer non-hemolytic antibodies to amustaline/GSH RBC were detected in clinical trials using a prior version of the process. The amustaline/GSH process was re-formulated to decrease S-303 RBC adduct formation. STUDY DESIGN AND METHODS: A standard three-cell antibody screening panel was modified to include reagent red cells (RRC) with high (S-303H) or low (S-303L) S-303 adduct density as assessed by flow cytometry, representative of the original and current amustaline/GSH treatment processes, respectively. General hospital and RBC transfusion-dependent patients never exposed, and clinical trial subjects exposed to amustaline/GSH RBC were screened for antibodies to amustaline/GSH RBC using a standardized agglutination assay. RESULTS: Twelve (0.1%) of 10,721 general hospital and 5 (0.5%) of 998 repeatedly-transfused patients not previously exposed to amustaline/GSH RBCs expressed natural, low titer (2-32) IgM and/or IgG (non-IgG1 or IgG3 isotype) antibodies with acridine (a structural element of amustaline) (n = 14) or non-acridine (n = 3) specificity. 11 of 17 sera reacted with S-303L panel RRCs. In clinical studies 81 thalassemia and 25 cardiac surgery patients were transfused with a total of 1085 amustaline/GSH RBCs and no natural or treatment-emergent S-303 antibodies were detected. CONCLUSION: Standardized RRC screening panels are sensitive for the detection of natural and acquired S-303-specific antibodies. Natural low titer antibodies to amustaline/GSH RBC are present in 0.15% of naïve patients. The clinical relevance of these antibodies appears minimal but is under further investigation.

Detection of SARS-CoV-2 neutralizing antibodies with a cell-free PCR assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 4 million confirmed infections worldwide and over 300,000 deaths. While Remdesivir has recently received FDA emergency use authorization for treatment of SARS-CoV-2 infection, convalescent plasma (CP) with high titers of SARS-CoV-2 neutralizing antibodies (NAbs) from recovered donors remains a promising and widely accessible method to mitigate severe disease symptoms. Here, we describe the development and validation of a cell-free neutralization PCR assay using SARS-CoV-2 spike protein S1 and human ACE2 receptor-DNA conjugates. By comparing with samples collected prior to the outbreak, we confirmed that NAbs were specifically detected in COVID-19 cases. Using our unique assay, the NAb signals are detectable as early as 10 days after onset of symptoms and continue to rise, plateauing after 18 days. Notably, we showed that the use of licensed pathogen reduction technology to inactivate potentially contaminating infectious pathogens in CP did not alter NAb signals, paving a path to safely administer effective CP therapies. The described neutralization PCR assay can serve as a qualification tool to easily identify suitable CP donors of a potentially lifesaving therapy. In addition, this assay tool is readily deployable in standard laboratories with biosafety level 2 capability, and can yield results within 2-3 hr. This advancement can facilitate research on factors driving diverse COVID-19 disease manifestations, and to evaluate the impact of various CP processing protocols on CP therapeutic efficacy.