Pathogen inactivation of blood components: Year in a review

1. Pathogen inactivation of blood components: Year in a review Dr. Anju Dubey

2. Introduction • Pathogen inactivation is a technology which calls for addition of various additives to blood product to inactivate viruses, bacteria, protozoa and other transfusion transmitted infections. • Pathogen inactivation was the original term for the technology, but as it is argued that the inactivation may not be complete, some authors prefer the term “pathogen reduction”. • represents a proactive approach to blood safety. • promises an additional layer of protection from infectious agents that are known and from those not yet recognized as threats to the blood supply.

3. Rationale minimization of an already extremely low risk of viral transmission (residual infection) further reduction, but not total elimination, of the risk of transfusion-transmitted bacterial sepsis additional protection against cell-associated viruses, such as CMV Reduction of emerging and unknown pathogens. Reduction of prions. probable elimination of transfusion- associated GVHD possible attenuation of the immunomodulatory effects of transfusion

4. BLOOD Labile products Cellular Non labile products Acellular • RBC • DMMB • FRALE • Inactine • Porphyrins • Cyanines • Phenobarbitone • derivatives • Aziridine • FFP • SD • MBLT • PUVA • Riboflavin • Plasma derivatives • Fractionation • Pasteurization • Dry heat • Pressure cycling • Low pH • Platelets • PUVA • Riboflavin • Thionine

5. Current pathogen inactivation techniques

6. Current inactivation technologies

7. Important studies in last one year

8. Generation of procoagulant collagen‐ and thrombin‐activated platelets in platelet concentrates derived from buffy coat: the role of processing, pathogen inactivation, and storage • Collagen and thrombin-activated (COAT) platelets, generated by dual-agonist stimulation with collagen and thrombin (THR), enhance THR generation at the site of vessel wall injury. A decreased ability to generate them is associated with bleeding diathesis. • Aim was to study PLT functions, particularly the ability to generate COAT PLTs, in PLT concentrates pathogen inactivation treatment (amotosalen-UVA). • Two PCs from five donors each were pooled and split in two bags; one of them was pathogen inactivated and the other one was left untreated. Flow cytometric analyses were performed immediately after PC preparation (Day 1) and thereafter on Days 2, 5, 7, and 9 in treated and untreated PCs to measure the reactivity of PLTs (CD62P and PAC- 1), the content and secretion of dense granule after stimulation with different agonists, and the percentage of COAT PLTs after dual stimulation with convulxinand THR. • Preparation of PCs by buffy coat method resulted in a significant decrease of COAT PLTs and in an impaired response to adenosine 50-diphosphate sodium (ADP). • Minor differences were observed between untreated or amotosalen-UVA–treated PCs. TRANSFUSION 2018;58;2395–2406

9. Ultraviolet light-based pathogen inactivation and alloimmunization after platelet transfusion: results from a randomized trial • The study explored whether pathogen-reduction treatment of platelet components before transfusion would decrease the risk of alloimmunization. • Study participants were patients with hematologic cancer who were included in two parallel, randomized clinical trials testing pathogen reduction treatment versus conventional platelets using the Mirasol or Intercept pathogen-reduction systems. Patients who had a baseline, pretransfusion sample and a follow-up, posttransfusion sample were included in the study. • Human leukocyte antigen antibody levels were determined using a commercial multianalyte, bead-based assay. • The rate of human leukocyte antigen Class I alloimmunization at the clinical sites in recipients of conventional platelets was low at the highest assay cut-off. Consistent with prior studies, human leukocyte antigen antibodies were first detected from 3 to 35 days after transfusion. • There were no statistically significant differences between alloimmunization rates in patients who received pathogen-reduction treatment versus conventional platelet transfusions. TRANSFUSION 2018;58;1210–1217

10. Mitochondrial DNA multiplex real-time PCR inhibition assay for quality control of pathogen inactivation by UV C light in platelet concentrates • Amotosalen/UVA and riboflavin/UVbased PI technologies induce modifications of the PLTderived mitochondrial DNA that can be detected by PCR inhibition assays. • This study sought to establish a PCR inhibition assay to document the impact of UVC treatment with the THERAFLEX UV-Platelets system on the mitochondrial genome in PLT concentrates. • A multiplex realtime PCR inhibition assay with simultaneous shortamplicon (143 bp) and long-amplicon (794 bp) amplification was developed to detect mtDNA modifications in PLTs after UVC treatment. Assay performance was tested in UVC-treated and untreated, plasma-reduced pooled PCs, and apheresis PCs. • UVC illumination of PLTs resulted in dose dependent inhibition of mtDNA amplification for the larger amplicon. Amplification of the shorter amplicon was not affected by UVC treatment. Evaluation of 283 blinded apheresis and pooled PLT samples from routine-like PC production resulted in prediction of UVC treatment status with 100% accuracy. • The proposed dual-amplicon size realtime mtDNA PCR assay effectively detects nucleic acid damage induced by UVC illumination of PLTs and could be useful as an informative indicator of PI quality of the THERAFLEX UV-Platelets system. TRANSFUSION 2018;58;758–765

11. Cryopreservation of buffy coat–derived platelet concentrates photochemically treated with amotosalen and UVA light • The objective of this study was to analyze potential effects of PCT on CPPs as compared with untreated CPPs. • A total of 12 PCT treated and 12 untreated platelet units from buffy coats were cryopreserved at −80C in 5% dimethyl sulfoxide. CPPs of both types were rapidly thawed at 37C and resuspended in 200 mL fresh plasma. In vitro properties were analyzed prefreezing, postfreezing and thawing, and on Day 1 after thawing. • Directly after thawing, no major differences in platelet content, lactase hydrogenase, adenosine triphosphate, mitochondrial membrane potential, CD62P, CD42b, and platelet endothelial cell adhesion molecule were seen between PCT-CPPs and conventional CPPs. • On Day 1 after thawing, the CPPs of both types performed less well. The PCT-CPPs tended to be more affected by the freezing process than the conventional CPPs. • PCT-CPPs appeared slightly more susceptible to lesion effects by freezing than conventional CPPs, in particular in assays on Day 1 after thawing, but these differences were small relative to the dramatic effects of the freezing process itself. TRANSFUSION 2018;58;1–12

12. Plasma temperature during methylene blue/light treatment influences virus inactivation capacity and product quality • The influence of different temperature conditions on virus inactivation capacity and plasma quality of the THERAFLEX MB-Plasma procedure was investigated in this study. • Plasma units equilibrated to 5°C, room temperature 22°C or 30 °C were treated with MB/light and comparatively assessed for the inactivation capacity for three different viruses, concentrations of MB and its photoproducts, activity of various plasma coagulation factors and clotting time. • Reduced solubility of the MB pill was observed at 5 – 2°C. Photocatalytic degradation of MB increased with increasing temperature, and the greatest formation of photoproducts (mainly azure B) occurred at 30 °C. Inactivation of herpesvirus, bovine viral diarrhoea virus and vesicular stomatitis virus was significantly lower at 5°C than at higher temperatures. • Increasing plasma temperatures resulted in greater changes in clotting time and higher losses of plasma coagulation factor activity. • Temperature conditions for THERAFLEX MB-Plasma treatment must be carefully controlled to assure uniform quality of pathogen-reduced plasma in routine production. Inactivation of cooled plasma is not recommended. VOX SANGUINIS 2018, 113: 368–377

13. Effective inactivation of a wide range of viruses by pasteurization • Pasteurization at 60°C for 10 hours have been implemented in the manufacturing process of therapeutic plasma proteins such as human albumin, coagulation factors, immunoglobulins, and enzyme inhibitors to inactivate blood‐borne viruses of concern.  • The virus inactivation kinetics of pasteurization for a broad range of viruses were evaluated in the relevant intermediates from more than 15 different plasma manufacturing processes.  • The data demonstrate that pasteurization inactivates a wide range of enveloped and nonenveloped viruses of diverse physicochemical characteristics. After a maximum of 6 hours' incubation, no residual infectivity could be detected for the majority of enveloped viruses. Effective inactivation of a range of nonenveloped viruses, with the exception of nonhuman parvoviruses, was documented. • Pasteurization is a very robust and reliable virus inactivation method with a broad effectiveness against known blood‐borne pathogens and emerging or potentially emerging viruses. • Pasteurization has proven itself to be a highly effective step, in combination with other complementary safety measures, toward assuring the virus safety of final product. TRANSFUSION 2018;58;41–51

14. Inactivation of Middle East respiratory syndrome‐coronavirus in human plasma using amotosalen and ultraviolet A light • Study investigated the efficacy of amotosalen and UVA to inactivate MERS-CoV in FFP • Pooled FFP units were spiked with a recent clinical MERS-CoVisolate. Infectious and genomic viral titers were determined in plasma before and after inactivation with amotosalen/ UVA treatment by plaque assay and reverse transcription–quantitative polymerase chain reaction, respectively. • In addition, residual replicating or live virus after inactivation was examined by passaging in the permissive Vero E6 cells. • The mean MERS-CoV infectious titerin pretreatmentsamples was reduced to undetectable levels after inactivation with amotosalen/ UVA. • Furthermore, inoculation of inactivated plasma on Vero E6 cells did not result in any cytopathiceffect even after 7 days of incubation nor the detection of MERS RNA compared to pretreatmentsamples. • Data show that amotosalen/UVA treatment is a potent and effective way to inactivate MERS-CoVinfectious particles in FFP to undetectable levels and to minimize the risk of any possible transfusion-related MERS-CoV transmission TRANSFUSION 2018;58;52–59

15. Inactivation of chikungunya virus in blood components treated with amotosalen/ultraviolet A light or amustaline/glutathione • Inactivation of chikungunya virus was investigated for platelets in 100% plasma using amotosalen/ultraviolet A light, and in red blood cells using amustaline/glutathione. • Platelets and RBCs were spiked with chikungunya virus. Infectious chikungunya virus titers were measured in contaminated blood products before and after treatment with amotosalen/ultraviolet A light for platelets and after treatment with amustaline/glutathione for RBCs. Viral infectivity was quantified by plaque assay. • The mean chikungunya virus infectivity titers before inactivation were 6.50 log10 plaque-forming units/ mL for platelets in 100% plasma and 7.60 log10 plaque forming units/mL for RBCs. No infectivity was detected after amotosalen/ultraviolet A light or amustaline/ glutathione treatment. • Robust levels of chikungunya virus inactivation were achieved for platelets for RBC components. • The licensed amotosalen/ ultraviolet A light technology and the amustaline/glutathione pathogen-reduction system under development may provide an opportunity for comprehensive mitigation of the risk of chikungunya virus transfusion-transmitted infection by plasma, platelets, and RBCs. TRANSFUSION 2018;58;748–757

16. Inactivation of Ebola virus and Middle East respiratory syndrome coronavirus in platelet concentrates and plasma by ultraviolet C light and methylene blue plus visible light, respectively • This study investigated the efficacy of the THERAFLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation systems to inactivate EBOV and MERS-CoV in platelet concentrates and plasma, respectively. • PCs and plasma were spiked with high titers of cell culture–derived EBOV and MERS-CoV, treated with various light doses of ultraviolet C (UVC; THERAFLEX UV-Platelets) or methylene blue (MB) plus visible light (MB/light; THERAFLEX MB-Plasma), and assessed for residual viral infectivity. • UVC reduced EBOV (4.5 log) and MERS-CoV (3.7 log) infectivity in PCs to the limit of detection, and MB/light decreased EBOV (4.6 log) and MERS-CoV (3.3 log) titers in plasma to non-detectable levels. • Both THERAFLEX UV-Platelets (UVC) and THERAFLEX MB-Plasma (MB/light) effectively reduce EBOV and MERS-CoV infectivity in platelets and plasma, respectively TRANSFUSION 2018;58;2202–2207

17. Ultraviolet C light efficiently inactivates nonenveloped hepatitis Avirus and feline calicivirus in platelet concentrates • This study investigated the pathogen inactivation efficacy of the THERAFLEX UV Platelets system against two nonenveloped viruses: HAV and feline calicivirus (FCV), in platelet concentrates (PCs). • PCs in additive solution were spiked with high titers of cell culture– derived HAV and FCV, and treated with ultraviolet C at various doses. • Pre- and posttreatment samples were taken and the level of viral infectivity determined at each dose. For some samples, large-volume plating was performed to improve the detection limit of the virus assay. • THERAFLEX UV-Platelets reduced HAV titers in PCs to the limit of detection, resulting in a virus reduction factor of greater than 4.2 log steps, and reduced FCV infectivity in PCs by 3.0 log steps. • THERAFLEX UV-Platelets effectively inactivates HAV and FCV in platelet units. TRANSFUSION 2018;58;1–6

18. Amustaline (S-303) treatment inactivates high levels ofChikungunya virus in red-blood-cell components • The study evaluated the efficacy of amustaline and glutathione (S-303/GSH) to inactivate CHIKV in RBCs. • Red-blood-cells were spiked with high level of CHIKV. Infectious titres and RNA loads were measured before and after PI treatment. Residual CHIKV infectivity was also assessed after five successive cell culture passages. • No CHIKV TCID was detected after S-303 treatment nor was replicative CHIKV particles and viral RNA present after five cell culture passages of samples obtained immediately after S303 treatment. • Chikungunya virus was previously shown to be inactivated by the PI technology using amotosalen and ultraviolet A light for the treatment of plasma and platelets. • This study demonstrates that S-303 can inactivate high titres of CHIKV in RBCs. VOX SANGUINIS 2018; 113: 232–241

19. In vitro quality of amotosalen-UVA pathogen-inactivated mini-pool plasma prepared from whole blood stored overnight • The study evaluated the conservation of coagulation factors in AUVA-plasma prepared from WB stored overnight to determine its therapeutic efficacy. Thrombin generation by the AUVA-plasma was used to provide an integrated measure of the hemostatic capacity. • WB-donations stored overnight were processed to prepare leucocyte-depleted plasma mini-pools, which were divided into two parts and treated with AUVA. Each mini-pool yielded six AUVA-plasma units (200 ml) which were frozen (-25°C) within 19 h of WB-collection. Their hemostatic quality was evaluated before and after treatment for up to 12 months of storage. • Immediately after AUVA-treatment, the regulatory criteria for FVIII activity and fibrinogen content were met. As compared to untreated plasma there was a reduction in fibrinogen (14%), FV (9%), FVII (25%) and FVIII (32%). However, Thrombin generation was similar in treated and untreated plasma at all-time-points. • Frozen WB-derived AUVA-plasma prepared from mini-pools within 19 h of WB-collection met the quality standards required and retained hemostatic capacity for up to 12 months. This product could provide a cost effective convenient substitute for apheresis plasma. VOX SANGUINIS 2018; 113: 622–631

20. Amotosalen/UVA treatment inactivates T cells more effectively than the recommended gamma dose for prevention of TAGVHD • In this study, T-cell inactivation was compared, between amotosalen/ UVA treatment and gamma-irradiation (2500 cGy), using a sensitive limiting dilution assay with an enhanced dynamic range. • Matched plasma units, contaminated with peripheral blood mononuclear cells, were either treated with amotosalen/UVA or gamma irradiation, or retained as untreated control. • Posttreatment, cells were cultured under standardized conditions. T-cell proliferation was determined by the incorporation of 3 H-thymidine and correlated with microscopic detection. • Complete inactivation of the T cells after amotosalen/UVA treatment was observed, equivalent to greater than 6.2 log inactivation. • Amotosalen/UVA treatment more effectively inactivates T cells than the current standard of gamma irradiation (2500 cGy) for the prevention of TA-GVHD TRANSFUSION 2018;58;1506–1515

21. TA-GVHD re-examined: potential for improved prevention using a universally applied intervention • TA‐GVHD clinical case literature including numerous original clinical case reports and several comprehensive case series were examined. Also evaluated recent in vitro experimental data on the inhibition of T cell proliferation, comparing the effect of a specific pathogen inactivation technology to that of the Food and Drug Administration–recommended gamma irradiation dose of 2500 cGy. • Identified 12 published TA‐GVHD cases with atypical/milder or delayed symptom presentations and/or an atypical clinical course; these included cases attributed to leukoreduced or suboptimally irradiated units. Summarize recent in vitro data using a sensitive limiting dilution assay that establish that, compared to irradiation at the recommended 2500 cGy dose, PI using amotosalen/ultraviolet A, or amustaline/glutathione achieves a greater degree of inhibition of T cell proliferation. • PI technologies appear to be at least equal to, if not better than, gamma irradiation in abrogating the ability of T cells to proliferate, and if applied to all blood components, protection against TA‐GVHD would be an additional benefit that would allow for the elimination of component irradiation. TRANSFUSION 2018;58;2545–2563

22. Red blood cells treated with the amustaline (S‐303) pathogen reduction system: a transfusion study in cardiac surgery • A randomized, double‐blind, controlled study was performed to assess the in vitro characteristics of amustaline‐treated RBCs (test) compared with conventional (control) RBCs and to evaluate safety and efficacy of transfusion during and after cardiac surgery. • A total of 774 RBC units were produced. Amustaline‐treated RBCs met European guidelines for Hb content, hematocrit, and hemolysis. Fifty‐one (25 test and 26 control) patients received study RBCs. • There were no significant differences in RBC usage or other clinical outcomes. Observed AEs were within the spectrum expected for patients of similar age undergoing cardiovascular surgery requiring RBCs transfusion. No patients exhibited an immune response specific to amustaline‐treated RBCs • Amustaline‐treated RBCs demonstrated equivalence to control RBCs for Hb content, have appropriate characteristics for transfusion, and were well tolerated when transfused in support of acute anemia. TRANSFUSION 2018;58;905–916

23. Nationwide Implementation of Pathogen Inactivation for All Platelet Concentrates in Switzerland • Based on numbers of transfused units and mandatorily reported adverse events with high imputability, the study compare the risks associated with transfusion of conventional PCs and pathogen-inactivated PCs • From 2005 to 2011, a total of 158,502 cPCs have been issued in Switzerland, and 16 transfusion-transmitted bacterial infections (including 3 fatalities) were reported. • From 2011 to 2016, a total of 205,574 PI-PCs have been issued, and no transfusion-transmitted bacterial infection was reported. • Observed reductions of 66% for life-threatening and fatal reactions and of 26% for all high-imputability transfusion reactions related to PI-PCs as compared to cPCs. No increased rates of bleeding or clinical observations of ineffectiveness of PI-PCs have been reported. • Swiss hemovigilance data confirm a favorable safety profile of the nationwide introduced Intercept pathogen inactivation procedure and its reliable prevention of septic transfusion reactions and fatalities due to bacterially contaminated PCs. TRANSFUS MED HEMOTHER 2018;45:151-156

24. Biomolecular Consequences of Platelet Pathogen Inactivation Methods TRANSFUS MED REV 2018 JUN 27

25. Take home message • PRTs are an additional safety measure to prevent transfusion transmission of pathogens. • They provide the ability to inactivate levels of both tested and untested pathogens that can sometimes be present at very low concentration in blood components. • Understanding pathogen dynamics during infection, measuring the number of infectious particles in blood products, and determining the transfusion-transmission relevance of a given pathogen is important to assess a PRT’s effectiveness at reducing the risk of disease transmission. • Such evaluations should balance the effectiveness of a technology in reducing pathogen risk with equally important considerations such as the toxicological and clinical effectiveness implications of treatment to reduce pathogen load and how these may subsequently impact therapeutic efficacy. • Only when considering all of these issues can a correctly balanced and fully informed decision be made.

26. Thank you Pathogen Inactivation Represents a New Paradigm Shift for Transfusion Medicine