TnBP⁄Triton X-45 treatment of plasma for transfusion efficiently inactivates hepatitis C virus

Abstract

Risk of transmission of hepatitis C virus (HCV) by clinical plasma remains high in countries with a high prevalence of hepatitis C, justifying the implementation of viral inactivation treatments. In this study, we assessed the extent of inactivation of HCV during minipool solvent/detergent (SD; 1% TnBP / 1% Triton X-45) treatment of human plasma. Luciferase-tagged infectious cell culture-derived HCV (HCVcc) particles were used to spike human plasma prior to treatment by SD at 31 ± 0.5°C for 30 min. Samples were taken before and after SD treatment and filtered on a Sep-Pak Plus C18 cartridge to remove the SD agents. Risk of cytotoxicity was assessed by XTT cell viability assay. Viral infectivity was analyzed based on the luciferase signals, 50% tissue culture infectious dose viral titer, and immunofluorescence staining for HCV NS5A protein. Total protein, cholesterol, and triglyceride contents were determined before and after SD treatment and C18 cartridge filtration. Binding analysis, using patient-derived HCV clinical isolates, was also examined to validate the efficacy of the inactivation by SD. SD treatment effectively inactivated HCVcc within 30 min, as demonstrated by the baseline level of reporter signals, total loss of viral infectivity, and absence of viral protein NS5A. SD specifically targeted HCV particles to render them inactive, with essentially no effect on plasma protein content and hemostatic function. More importantly, the efficacy of the SD inactivation method was confirmed against various genotypes of patient-derived HCV clinical isolates and against HCVcc infection of primary human hepatocytes. Therefore, treatment by 1% TnBP / 1% Triton X-45 at 31°C is highly efficient to inactivate HCV in plasma for transfusion, showing its capacity to enhance the safety of therapeutic plasma products. We propose that the methodology used here to study HCV infectivity can be valuable in the validation of viral inactivation and removal processes of human plasma-derived products.

Fig 1. Flowchart of SD treatment and C18 filtration procedure on human plasma for HCV infectivity assay.

Fig 2. C18 efficiently removes SD cytotoxicity.

Plasma and SD/C18 treatments were tested for potential cytotoxicity. Huh-7.5 cells seeded at sub-confluence in 96-well plates (1 × 10⁴ cells per well) were incubated in various treatments for 72 h before analysis by XTT cell viability assay. Data shown are collected from three independent experiments with means ± SEM (***: P < 0.001). Plasma: virus-free plasma; PBS/SD: PBS treated with SD (1% TnBP / 1% Triton X-45); Plasma + SD/C18: C18 cartridge filtration of plasma treated with SD.

Fig 3. SD directly inactivates HCV particles.

To test the specificity of SD in inactivating HCV particles, cell-free virus was co-incubated with low dose SD (0.01%, 0.05%) for 3 h and then diluted 50× to sub-cytotoxic concentrations of the treatment before infecting Huh-7.5 cells (MOI = 0.01). Viral infectivity was measured by luciferase reporter activity (RLU). Data shown are collected from three independent experiments with means ± SEM (*: P < 0.05).

Fig 4. SD treatment efficiently removes HCV infectivity from virus-containing plasma.

Infectivity of HCV-spiked plasma (Plasma_HCV) with or without SD treatment followed by C18 filtration (SD/C18) was analyzed by: A. Immunofluorescence staining of viral NS5A (magnification: 100×; scale bar = 100 μm); B. Virus titer determination by TCID₅₀ analysis (units/ml); and C. Luciferase reporter measurement of viral infectivity (RLU). All quantifiable data and representative micrographs are from three independent experiments; error bars indicate means ± SEM (***: P < 0.001). See text for details.

Fig 5. SD treatment efficiently inactivates clinical HCV isolates.

Patient-derived HCV particles of various genotypes were treated with or without SD/C18 procedure and then used to challenge Huh-7.5 cells. Total HCV RNA on Huh-7.5 cell surface was subsequently extracted and quantified by COBAS AMPLICOR HCV MONITOR test. Mean values from duplicate experiments are shown (IU/ml).

Fig 6. HCVcc infection of primary human hepatocytes is abolished by SD treatment.

Plated primary human hepatocytes were challenged with plasmas only (Mock), HCVcc-spiked plasma (Plasma_HCV), or HCVcc-spiked plasma with SD treatment followed by C18 filtration (Plasma_HCV + SD/C18), then washed, and subsequently analyzed for luciferase activity (RLU) in the supernatant to quantify viral infectivity following 5 days of incubation in primary hepatocyte culturing medium. Error bars indicate means ± SEM from three independent experiments (***: P < 0.001). See text for details.