Amustaline (S‐303) treatment inactivates high levels of Zika virus in red blood cell components

Abstract

BACKGROUND:The potential for Zika virus (ZIKV) transfusion-transmission (TT) has been demonstrated in French Polynesia and Brazil. Pathogen inactivation (PI) of blood products is a proactive strategy to inactivate TT pathogens including arboviruses. Inactivation of West Nile, dengue, Zika, and chikungunya viruses was previously demonstrated by photochemical treatment with amotosalen and ultraviolet A (UVA) illumination. In this study, we evaluated ZIKV inactivation in red blood cell (RBC) components by a chemical approach that uses amustaline (S-303) and glutathione (GSH). STUDY DESIGN AND METHODS: RBC componentswere spiked with a high titer of ZIKV. Viral titers (infectivity) and ZIKV RNA loads (reverse transcriptionpolymerase chain reaction) were measured in spiked RBCs before and after S-303 and GSH treatment and confirmed using repetitive passages in cell culture. A mock-treated arm validated the approach by demonstrating stability of the virus (infectivity and RNA load) during the process. RESULTS:The mean ZIKV infectivity titer and RNA load in RBCs were 5.99 6 0.2 log 50% tissue culture infectious dose (TCID 50 )/mL and 7.75 6 0.16 log genomic equivalents/mL before inactivation. No infectivity was detected immediately after S-303 and GSH treatment and after five serial passages in cell culture. CONCLUSION:Complete ZIKV inactivation of more than 5.99 log TCID 50 /mL in RBCs was achieved using S-303 and GSH at levels higher than those found in asymptomatic ZIKV-infected blood donors. Therefore, the S-303 and GSH PI system is promising for mitigating the risk of ZIKV TT. US areas with active ZIKV circulation, the main recommendations included donor deferral, blood collection suspension, and importation of blood components from nonactive transmission areas and either implementation of ZIKV nucleic acid testing (NAT) or pathogen inactivation (PI). PI is a proactive strategy designed to reduce or eliminate infectivity of bacteria, viruses, and parasites in blood. 35 Several processes have been developed for the inactivation of pathogens in fresh-frozen plasma and platelet (PLT) concentrates. 35In FP, the INTERCEPT Blood System for PLTs (Cerus Corporation), a CE mark-approved technology, was implemented in 2010 and has since been used routinely. During the FP ZIKV outbreak, plasma units were imported from metropolitan France. 22 However, during the outbreak, no PI technology was commercially available for red blood cell (RBC) treatment.The INTERCEPT Blood System for RBCs uses a chemical treatment with the small molecule amustaline (S-303) that forms covalent crosslinks and adducts with nucleic acids resulting in the inactivation of pathogens. S-303 is a reactive modular compound that is designed to react quickly and then decompose by hydrolysis to the nonreactive compound S-300 (Fig. 1). It is formulated in the presence of the natural tripeptide glutathione (GSH) to quench unreacted side reactions. S-303 has been shown to inactivate a variety of enveloped and nonenveloped viruses, Gram-positive and -negative b...

Inactivation of chikungunya virus in blood components treated with amotosalen/ultraviolet A light or amustaline/glutathione

“…Our previous studies illustrated the ability of the amotosalen/UVA system to inactivate greater than 6.4 log 10 TCID 50 /mL in platelets in plasma-PAS, equating to greater than 8.7 log 10 GEq/mL of CHIKV RNA and greater than or equal to 7.6 log 10 TCID 50 / mL in plasma, corresponding to greater than 9.9 log 10 GEq/mL of CHIKV RNA (Table 3). 33 These results are consistent with similar correlations identified in other arboviruses, such as Zika, 36 dengue 40 and West Nile viruses, 41 in which the genomic viral loads correspond from 100-fold to 1000-fold the infectivity titers observed in the same samples, as determined by comparing infectivity titers measured by cell culture assays with viral loads measured by nucleic acid amplification assays. With CHIKV and other arboviruses emerging and reemerging in various geographies, the safety of the blood supply is at stake in both endemic and nonendemic areas.…”

“…B). The RBC units were treated with amustaline/GSH, as described by Laughhunn and colleagues . Two preinactivation samples were taken from all units: one was frozen immediately after inoculation with CHIKV and dosing with GSH, and the other was allowed to incubate at room temperature for 3 hours to mirror the treated RBC units.…”

“…The RBC units were treated with amustaline/GSH, as described by Laughhunn and colleagues. 36 Two preinactivation samples were taken from all units: one was frozen immediately after inoculation with CHIKV and dosing with GSH, and the other was allowed to incubate at room temperature for 3 hours to mirror the treated RBC units. After removal of the control samples, the RBC units were dosed with amustaline and then transferred to the incubation container and stored at room temperature for 3 hours.…”

Reduction of Zika virus infectivity in platelet concentrates after treatment with ultraviolet C light and in plasma after treatment with methylene blue and visible light

“…Inactivation of ZIKV with the INTERCEPT PI system in PLTs suspended in 35 or 100% plasma resulted in more than 4.1 log reduction in viral infectivity, whereas for plasma, more than 6.46 log reduction was achieved . Laughhunn and colleagues have also shown the capacity of a novel inactivation process to reduce ZIKV infectivity in red blood cells (RBCs) by more than 4.3 log, whereby components are treated with amustaline (S‐303) and glutathione without the need for illumination. Variable viral loads have been detected in ZIKV‐infected blood donors, and not all recipients of ZIKV‐infected blood develop symptoms indicative of disease after transfusion .…”

Inactivation of Zika virus in platelet components using amotosalen and ultraviolet A illumination

“…With the completion of this study, it has now been formally demonstrated that nucleic acid-targeted PI methodologies effectively inactivate ZIKV in all three blood components: in plasma (>6.57 log 10 TCID 50 , >10.25 log 10-GEq/mL) 28 and platelets (>4.42 log 10 TCID 50 , >7.46 log 10 GEq/mL) using amotosalen/UVA and in red blood cells (>5.99 log 10 TCID 50 , 7.75 log 10 GEq/mL) using amustaline/ glutathione 31 (Table 6). In addition, similar levels of inactivation have been recorded for other common arboviruses, such as CHIKV, DENV, and WNV (Table 6).…”