Amotosalen and ultraviolet A light treatment efficiently inactivates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human plasma

Abstract

Background and objectives: During the ongoing pandemic of COVID-19, SARS-CoV-2 RNA was detected in plasma and platelet products from asymptomatic blood donors, raising concerns about potential risk of transfusion transmission, also in the context of the current therapeutic approach utilizing plasma from convalescent donors. The objective of this study was to assess the efficacy of amotosalen/UVA light treatment to inactivate SARS-CoV-2 in human plasma to reduce the risk of potential transmission through blood transfusion.

Methods: Pools of three whole-blood-derived human plasma units (630-650 ml) were inoculated with a clinical SARS-CoV-2 isolate. Spiked units were treated with amotosalen/UVA light (INTERCEPT Blood System™) to inactivate SARS-CoV-2. Infectious titres and genomic viral load were assessed by plaque assay and real-time quantitative PCR. Inactivated samples were subject to three successive passages on permissive tissue culture to exclude the presence of replication-competent viral particles.

Results: Inactivation of infectious viral particles in spiked plasma units below the limit of detection was achieved by amotosalen/UVA light treatment with a mean log reduction of >3·32 ± 0·2. Passaging of inactivated samples on permissive tissue showed no viral replication even after 9 days of incubation and three passages, confirming complete inactivation. The treatment also inhibited NAT detection by nucleic acid modification with a mean log reduction of 2·92 ± 0·87 PFU genomic equivalents.

Conclusion: Amotosalen/UVA light treatment of SARS-CoV-2 spiked human plasma units efficiently and completely inactivated >3·32 ± 0·2 log of SARS-CoV-2 infectivity, showing that such treatment could minimize the risk of transfusion-related SARS-CoV-2 transmission.

Keywords: SARS-CoV-2; amotosalen/UVA; pathogen inactivation; plasma.

Figure 1

Experimental design. Schematic view of the experimental design. Briefly, pools of 3 plasma units were spiked in a 1:100 dilution with a SARS‐CoV‐2 viral stock. The pools were treated with amotosalen/UVA pathogen inactivation and post‐inactivation subject to 3 consecutive passages of 3 days respectively on Vero E6 cells. Samples were taken at various stages and assessed for replicating virus by plaque assay and viral genomes by RT‐qPCR.

Figure 2

Inactivation of SARS‐CoV‐2 in plasma by amotosalen and UVA treatment assessed by a plaque assay. Vero E6 cells were inoculated for 1 h with the following samples in a 1:10 dilution in DMEM: the SARS‐CoV‐2 viral stock (Positive Control), human plasma (Negative Control), plasma from a SARS‐CoV‐2 spiked pretreatment sample (Spiked Plasma Unit) and amotosalen/UVA‐treated, SARS‐CoV‐2 spikes plasma (Post‐iInactivation). The cells were overlaid with agarose, incubated for three more days followed by neutral red staining. Experiments were conducted in serial dilutions. Photographs (4×) are shown from one of five representative experiments.

Figure 3

Assessment of complete inactivation of replicative SARS‐CoV‐2 post‐amotosalen/UVA treatment by passaging experiments. Vero E6 cells were inoculated for 1 h with the following samples in a 1:10 dilution in DMEM: plasma from a SARS‐CoV‐2 spiked pretreatment sample (Positive Control), human plasma (Negative Control) and amotosalen/UVA‐treated, SARS‐CoV‐2 spikes plasma (Passage 1–3) and passaged for three consecutive passages. Both the positive control and the pretreatment sample caused extensive CPE by day 3 post‐inoculation in all three passages. Negative control and inactivated sample did not show any CPE in Vero E6 cells. Photographs (4×) are shown from one of five representative experiments on day 3 post‐inoculation in each passage.