A novel competition ELISA for the rapid quantification of SARS-CoV-2 neutralizing antibodies in convalescent plasma.

BACKGROUND: COVID-19 convalescent plasma (CCP) ideally contains high titers of (neutralizing) anti-SARS-CoV-2 antibodies. Several scalable immunoassays for CCP selection have been developed. We designed an enzyme-linked immunosorbent assay (ELISA) that measures neutralizing antibodies (of all isotypes) in plasma by determining the level of competition between CCP and a mouse neutralizing antibody for binding to the receptor binding domain (RBD) of SARS-CoV-2. METHODS: Plasma was collected from 72 convalescent individuals and inhibition of viral infection was determined by plaque reduction neutralization (PRNT50). The level of neutralizing antibodies was measured in the novel competition ELISA and in a commercially available ELISA that measures inhibition of recombinant ACE2 binding to immobilized RBD. These results were compared with a high throughput chemiluminescent microparticle immunoassay (CMIA). RESULTS: The results from both ELISAs were correlating, in particular for high titer CCP (PRNT50 ≥ 1:160) (Spearman r = .73, p < .001). Moderate correlation was found between the competition ELISA and CMIA (r = .57 for high titer and r = .62 for low titer CCP, p < .001). Receiver operator characteristic analysis showed that the competition ELISA selected CCP with a sensitivity and specificity of 61% and 100%, respectively. However, discrimination between low and high titer CCP had a lower resolution (sensitivity: 34% and specificity: 89%). CONCLUSION: The competition ELISA screens for neutralizing antibodies in CCP by competition for just a single epitope. It exerts a sensitivity of 61% with no false identifications. These ELISA designs can be used for epitope mapping or for selection of CCP.

The senotherapeutic nicotinamide riboside raises platelet nicotinamide adenine dinucleotide levels but cannot prevent storage lesion.

BACKGROUND: Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide riboside (NR) has recently been shown to increase life-span of cells, tissues, and entire organisms. [Correction added on 13 December 2019, after first online publication: In the preceding sentence, "adenine nicotinamide" was revised to "nicotinamide adenine."] The impact of NR on platelet longevity has not been tested. STUDY DESIGN AND METHODS: A pool-and-split design of buffy coat derived platelet concentrates (PCs) was used. One arm was treated with cumulative doses of NR-triflate, the control arm with sodium triflate. Storage lesion was monitored for 23 days. Platelet metabolic and functional parameters were tested. Clearance of human platelets was measured in a mouse model of transfusion. RESULTS: Total intracellular NAD levels in platelets decreased two-fold from 4.8 ± 0.5 fmol (mean ± SD, n = 6) to 2.1 ± 1.8 fmol per 103 control cells, but increased almost 10-fold to 41.5 ± 4.1 fmol per 103 NR treated platelets. This high intracellular NAD level had no significant impact on platelet count, mean platelet volume, swirling, nor on lactate and glucose levels. Platelet aggregation and integrin αIIb ß3 activation declined steadily and comparably in both conditions. GPIbα levels were slightly lower in NR-treated platelets compared to control, but this was not caused by reduced receptor shedding because glycocalicin increased similarly. Apoptotic markers cytochrome c, Bcl-xL, cleaved caspase-3, and Bak were not different throughout storage for both conditions. Platelet survival in a mouse model of transfusion was not different between NR-treated and control platelets. CONCLUSION: Platelets carry the cellular machinery to metabolize NR into NAD at rates comparable to other eukaryotic cells. Unlike those cells, platelet life-span cannot be prolonged using this strategy.

Impact of cold storage on platelets treated with Intercept pathogen inactivation.

BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significantly affect PLT function. It is not known how PLTs function when both are combined. STUDY DESIGN AND METHODS: Standard PLT concentrates (PCs) were compared to pathogen-inactivated PCs treated with amotosalen photochemical treatment (AS-PCT) when stored at room (RT, 22°C), cold (4°C, n = 6), or cryopreservation (-80°C, n = 8) temperatures. The impact of alternative storage methods on both arms was studied in flow cytometry, light transmittance aggregometry, and hemostasis in collagen-coated microfluidic flow chambers. RESULTS: Platelet aggregation of cold-stored AS-PCT PLTs was 44% ± 11% compared to 57% ± 14% for cold-stored standard PLTs and 58% ± 21% for RT-stored AS-PCT PLTs. Integrin activation of cold-stored AS-PCT PLTs was 53% ± 9% compared to 77% ± 6% for cold-stored standard PLTs and 69% ± 13% for RT-stored AS-PCT PLTs. Coagulation of cold-stored AS-PCT PLTs started faster under flow (836 ± 140 sec) compared to cold-stored standard PLTs (960 ± 192 sec) and RT-stored AS-PCT PLTs (1134 ± 220 sec). Fibrin formation rate under flow was also highest for cold-stored AS-PCT PLTs. This was in line with thrombin generation in static conditions because cold-stored AS-PCT PLTs generated 297 ± 47 nmol/L thrombin compared to 159 ± 33 nmol/L for cold-stored standard PLTs and 83 ± 25 nmol/L for RT-stored AS-PCT PLTs. So despite decreased PLT activation and aggregation, cold storage of AS-PCT PLTs promoted coagulation. PLT aggregation of cryopreserved AS-PCT PLTs (23% ± 10%) was not significantly different from cryopreserved standard PLTs (25% ± 8%). CONCLUSION: This study shows that cold storage of AS-PCT PLTs further affects PLT activation and aggregation but promotes (pro)coagulation. Increased procoagulation was not observed after cryopreservation.

A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device.

BACKGROUND AND OBJECTIVES: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). MATERIALS AND METHODS: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. RESULTS: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0·0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 ± 9% versus 76 ± 11%, respectively (P < 0·0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7·4 platelet-like particles per input cell from PB compared to 4·2 from UCB (P = 0·02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. CONCLUSION: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.

Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol 3-Kinase Signal Transduction by Altering Plasma Membrane Packing.

Psoralen and ultraviolet A light (PUVA) are used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma, and graft-versus-host disease. DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery, they are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like amphipathic lipid-packing sensor and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton's tyrosine kinase effectors following activation of the collagen glycoprotein VI and thrombin protease-activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol 3,4,5-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (effective half-maximal concentration (EC50), 8.4 ± 1.1 versus 4.3 ± 1.1 µm) and glycoprotein VI (EC50, 1.61 ± 0.85 versus 0.26 ± 0.21 µg/ml) but not PAR4 (EC50, 50 ± 1 versus 58 ± 1 µm) signal transduction. Our findings were confirmed in T-cells from graft-versus-host disease patients treated with extracorporeal photopheresis, a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids, thereby inhibiting membrane recruitment of effector kinases.

Ultraviolet C light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro.

BACKGROUND: Ultraviolet (UV) light illumination in the presence of exogenously added photosensitizers has been used to inactivate pathogens in platelet (PLT) concentrates for some time. The THERAFLEX UV-C system, however, illuminates PLT concentrates with UV-C light without additional photoactive compounds. In this study residual PLT function is measured in a comprehensive paired analysis of UV-C-treated, gamma-irradiated, and untreated control PLT concentrates. STUDY DESIGN AND METHODS: A pool-and-split design was used with buffy coat-derived PLT concentrates in 65% SSP+ additive solution. Thrombus formation kinetics in microfluidic flow chambers onto immobilized collagen was investigated with real-time video microscopy. PLT aggregation, membrane markers, and cellular metabolism were determined concurrently. RESULTS: Compared to gamma-treated and untreated controls, UV-C treatment significantly affected thrombus formation rates on Days 5 and 7, not Day 2. PLT degranulation (P-selectin) and PLT apoptosis (annexin V binding) was slightly but significantly increased from Day 2 on. UV-C treatment moreover induced integrin αIIb ß3 conformational changes reminiscent of activation. However, subsequent integrin activation by either PAR1-activating hexapeptide (PAR1AP) or convulxin was unaffected. This was confirmed by PLT aggregation studies induced with collagen, PAR1AP, and ristocetin at two different agonist concentrations. Finally, UV-C slightly increased lactic acid production rates, resulting in significantly decreased pH on Days 5 and 7, but never dropped below 7.2. CONCLUSION: UV-C pathogen inactivation treatment slightly but significantly increases PLT activation markers but does not profoundly influence activatability nor aggregation. The treatment does, however, attenuate thrombus formation kinetics in vitro in microfluidic flow chambers, especially after storage.

Oral administration of the Salmonella Typhimurium vaccine strain Nal2/Rif9/Rtt to laying hens at day of hatch reduces shedding and caecal colonization of Salmonella 4,12:i:-, the monophasic variant of Salmonella Typhimurium.

A new monophasic variant of Salmonella Typhimurium, Salmonella enterica serotype 4,12:i:-, is rapidly emerging. This serotype is now considered to be among the 10 most common serovars isolated from humans in many countries in Europe and in the United States. The public health risk posed by these emerging monophasic Salmonella Typhimurium strains is considered comparable to that of classical Salmonella Typhimurium strains. The serotype 4,12:i:- is frequently isolated from pigs but also poultry are carrying strains from this serotype. In the current study, we evaluated the efficacy of the Salmonella Typhimurium strain Nal2/Rif9/Rtt, a strain contained in the commercially available live vaccines AviPro Salmonella Duo and AviPro Salmonella VacT, against infection with the emerging monophasic variant in poultry. Three independent trials were conducted. In all trials, laying type chicks were orally vaccinated with the Salmonella Typhimurium strain Nal2/Rif9/Rtt at d hatch, while the birds were challenged the next d with a different infection dose in each trial (low, high, and intermediate). For the intermediate-dose study, a seeder bird model was used in which one out of 3 animals were infected while all individual birds were infected in the other trials. Data obtained from each independent trial show that oral administration of the Salmonella Typhimurium strain Nal2/Rif9/Rtt at d hatch reduced shedding, caecal, and internal organ colonization of Salmonella Typhimurium 4,12:i:-, administered at d 2 life. This indicates that Salmonella Typhimurium strain Nal2/Rif9/Rtt can help to control Salmonella 4,12:i:- infections in poultry.

Intranasal administration of convalescent plasma protects against SARS-CoV-2 infection in hamsters.

BACKGROUND: Convalescent plasma (CP) transfusion is an early option for treating infections with pandemic potential, often preceding vaccine or antiviral drug rollout. Heterogenous findings from randomized clinical trials on transfusion of COVID-19 CP (CCP) have been reported. However, meta-analysis suggests that transfusion of high titer CCP is associated with a mortality benefit for COVID-19 outpatients or inpatients treated within 5 days after symptom onset, indicating the importance of early administration. METHODS: We tested if CCP is an effective prophylactic against SARS-CoV-2 infection by the intranasal administration of 25 µL CCP/nostril (i.e. 0.01-0.06 mg anti-RBD antibodies/kg) in hamsters exposed to infected littermates. FINDINGS: In this model, 40% of CCP treated hamsters were fully protected and 40% had significantly reduced viral loads, the remaining 20% was not protected. The effect seems dose-dependent because high-titer CCP from a vaccinated donor was more effective than low-titer CCP from a donation prior to vaccine rollout. Intranasal administration of human CCP resulted in a reactive (immune) response in hamster lungs, however this was not observed upon administration of hamster CCP. INTERPRETATION: We conclude that CCP is an effective prophylactic when used directly at the site of primary infection. This option should be considered in future prepandemic preparedness plans. FUNDING: Flanders Innovation & Entrepreneurship (VLAIO) and the Foundation for Scientific Research of the Belgian Red Cross Flanders.

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.