Inactivation of Pathogens in Red Blood Cells by a Modified Treatment Process Utilizing S-303 and Glutathione.
Authors: Sawyer L. , Mababangloob R. , Patel N. , Kinsey J. , Sampson-Johannes A. , Hanson D. 
Published: October 2019

Abstract
Background: Treatment of red blood cells (RBC) with 0.2 mM S-303 (an acridine compound) and 2.0 mM glutathione (GSH) has previously been shown to inactivate a variety of pathogens, including viruses, bacteria, protozoan parasites and contaminating leukocytes. During Phase III clinical trials antibodies to S-303-treated RBC were detected in a few trial participants. These antibodies were later determined to be specific to residual acridine bound to the RBC surface. As a result of these antibodies, a modification of the S-303 treatment process has been developed to reduce the potential immunogenicity of S-303-treated RBC. This modified process utilizes 0.2 mM S-303 and 20 mM GSH. In this abstract we report data from an on-going series of studies designed to evaluate the efficacy of bacterial and viral inactivation in RBC using the modified S-303 treatment process.
Methods: Leukoreduced RBC units of approximately 280 mL with a hematocrit of approximately 60% were prepared in AS-3 storage medium. RBC units were inoculated with approximately 6 logs/mL of organism when possible, and an aliquot was removed to serve as the untreated, input control. GSH in a solution of 2 equivalents NaOH was added to the inoculated units to a final concentration of 20 mM and mixed well. Following a 5 to 10-minute room temperature incubation, S-303 was added to a final concentration of 0.2 mM and the units were again mixed well and incubated at 20 to 25 °C for three hours. After incubation, samples were removed and assayed to detect residual viable organisms. Control preparations were titered immediately after preparation and again after the 3-hour incubation. Bacterial titers were determined by colony formation on agar plates and viral titers were determined by plaque formation in MT-2 cells (HIV), BT cells (BVDV), vero E6 cells (VSV) and A549 cells (Adenovirus). Bacteria were chosen to represent Gram positive and Gram negative organisms of relevance to transfusion. Vesicular stomatis virus (VSV) was used as a representative negative sense RNA virus, and Adenovirus 5 as a representative non-enveloped DNA virus. HIV and bovine viral diarrhea virus (BVDV, model for HCV) inactivation was evaluated because of their relevance to blood transfusion. Replicate experiments were performed for each organism and the results are expressed as the mean and standard deviation (SD) of the observed inactivation levels. Inactivation is expressed as log reduction.
Results: See table below. Both Gram positive and Gram negative bacteria were effectively inactivated by treatment with the modified S-303 process. This process also resulted in effective inactivation of both the enveloped and non-enveloped viruses tested.
Conclusion: The modified S-303 process effectively inactivates bacterial and viral pathogens in RBC.
Organism Log Reductiona (Mean ±SD) Staphylococcus aureus ≥6.9 ±0.4 Staphylococcus epidermidis ≥7.0 ±0.1 Yersinia enterocolitica 4.1 ±0.8 Escherichia coli 6.1 ±0.7 Serratia marcescens 4.0 ±0.4 HIV >5.8 ±0.2 BVDV >4.4 ±0.2 Vesicular stomatitis virus (VSV) 4.2 ±0.1 Adenovirus 5 ≥8.0 ±0.3