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

Chou ML, Burnouf T, Chang SP, Hung TC, Lin CC, Richardson CD, Lin LT - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0117800.g002: 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.

Mentions: We first carried out a cell viability assay using XTT to demonstrate that plasma alone and SD-treated plasma followed by C18 filtration (SD/C18) do not induce cytotoxicity. Treatment with plasma only was not cytotoxic to the Huh-7.5 cells, whereas treatment with the minipool SD (1% of TnBP / 1% of Triton X-45) produced, as expected, significant cell death. This cytotoxic effect from SD however could be completely removed by C18 filtration as shown in the SD-treated plasma group with C18 treatment (Fig. 2). Therefore, hydrophobic C18 filtration can efficiently remove SD that is present in the plasma sample. The reduced viability observed when using the SD-treated plasma, compared to untreated plasma, also suggests removal of some growth factors or hydrophobic nutrients during the SD procedure.


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

Chou ML, Burnouf T, Chang SP, Hung TC, Lin CC, Richardson CD, Lin LT - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320006&req=5

pone.0117800.g002: 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.
Mentions: We first carried out a cell viability assay using XTT to demonstrate that plasma alone and SD-treated plasma followed by C18 filtration (SD/C18) do not induce cytotoxicity. Treatment with plasma only was not cytotoxic to the Huh-7.5 cells, whereas treatment with the minipool SD (1% of TnBP / 1% of Triton X-45) produced, as expected, significant cell death. This cytotoxic effect from SD however could be completely removed by C18 filtration as shown in the SD-treated plasma group with C18 treatment (Fig. 2). Therefore, hydrophobic C18 filtration can efficiently remove SD that is present in the plasma sample. The reduced viability observed when using the SD-treated plasma, compared to untreated plasma, also suggests removal of some growth factors or hydrophobic nutrients during the SD procedure.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.

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.

No MeSH data available.


Related in: MedlinePlus