Inactivating hepatitis C virus in donor lungs using light therapies during normothermic ex vivo lung perfusion

Abstract

Availability of organs is a limiting factor for lung transplantation, leading to substantial mortality rates on the wait list. Use of organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), would increase organ donation, but these organs are generally not offered for transplantation due to a high risk of transmission. Here, we develop a method for treatment of HCV-infected human donor lungs that prevents HCV transmission. Physical viral clearance in combination with germicidal light-based therapies during normothermic ex-vivo Lung Perfusion (EVLP), a method for assessment and treatment of injured donor lungs, inactivates HCV virus in a short period of time. Such treatment is shown to be safe using a large animal EVLP-to-lung transplantation model. This strategy of treating viral infection in a donor organ during preservation could significantly increase the availability of organs for transplantation and encourages further clinical development.

Conflict of interest statement

M.Cypel, T.W., S.K. and M.L. are founders of XOR Labs Toronto and M.Cypel, T.W. and S.K are consultants for Lung Bioengineering. J.J.F is consultant for AbbVie, Gilead Sciences, Merck and ContraVir. The remaining authors declare no competing interests.

Figures

Fig. 1

The customized illumination device and its usage during ex-vivo lung perfusion (EVLP). a The apparatus depicted with a germicidal UVC lamp, which was designed to be used during EVLP allocated in sequence with other EVLP components, in a closed system. Mounted on a cylindric tube, the light source is inserted into a tubular quartz tube, surrounded by an opaque PVC tube, that prevents light from escaping from the illuminated cavity. b The EVLP system with the illumination device (irradiator). The lungs are placed into a specific organ chamber. The EVLP circuit is composed of a hard-shell reservoir, a leucocyte filter, a membrane oxygenator/heater and a centrifugal pump. The illumination device, conceived to per part of the EVLP circuit, interpolates the centrifugal pump and the pulmonary artery cannula. During EVLP, the perfusate is treated when illuminated in 360° during its passage

Fig. 2

Effect of EVLP and light-based therapies (LbT) on HCV RNA levels in HCV NAT + human donor lungs. a Paired study design: Lungs from same donor were separated to 2 distinct EVLP systems under different treatment conditions (n = 3, each): standard EVLP (control) vs. treatment (circuit exchange, UVC or PDT). b Effect of EVLP and associated treatments towards perfusate and lung tissue HCV levels measured by qPCR during 9 h of treatment. Lung tissue and EVLP perfusate measurement results were normalized for percentage of viral load decrease from baseline and presented as mean ± SEM. The two-way ANOVA statistical test was used for analysis. EVLP: ex vivo lung perfusion

Fig. 3

Infectivity studies (UVC) a Schematic illustration of the mini-EVLP circuit: roller pump, external heater and irradiator device. At the start of perfusion, 1.5 million/ml of HCV genotype 2a surrogate (JFH-1) was added to this circuit. b After several minutes of UV treatment in the mini-EVLP circuit, perfusate samples were collected and used for HCV quantification by qPCR. After 30 min of UVC irradiation the qPCR counts of HCV significantly decreased in all timepoints (n = 4, p < 0.001, one-way ANOVA), although virus RNA was still detectable after 180 min of treatment. Black dots represent qPCR results in IU/mL in the control group whereas red triangles represent the qPCR results in the UVC group. Centre line represents mean and bounds of box are standard deviation. c Quantification of infectivity loss using cluster counts. Immunofluorescence assessment of HCV infectivity using a hepatocyte cell line (Huh 7.5). Cells were double-stained with DAPI and HCV anti-core antibody, then infected hepatocytes clusters were counted. Infectivity rates significantly decreased after 15 min of irradiation (n = 4, p < 0.001, one-way ANOVA), demonstrating total infectivity loss after 150 min of treatment, in all 4 replicates used. Black dots represent cluster count results in the control group whereas red triangles represent cluster count in the UVC group. Centre line represents mean and bounds of box are standard deviation. d Representative immunofluorescence picture of infectivity loss in treated perfusate samples. Scale bar = 400 µm

Fig. 4

Infectivity studies (PDT). The perfusion solution in the mini-EVLP circuit was spiked with a set amount of JFH-1 viruses and hepatocyte Huh 7.5 were used as an infectivity assay (n = 4, each group). Methylene Blue (MB) was diluted in different concentrations (1 μM/L, 0.5 μM/L, 0.1 μM/L and 0.01 μM/L) and activated by different light conditions (no light, 660 nm/20 mW/cm² red light and regular room light). A Huh 7.5 hepatocyte cell culture was transfected and stained (DAPI and HCV anti-core Ab). Infected hepatocytes clusters were counted. a In dark conditions, HCV maintained the infectivity potential, regardless the MB dosage. qPCR counts of HCV were stable during the treatment. b Room light exposure promoted virus inactivation in a MB dose-dependent manner (p < 0.001 in all scenarios, one-way ANOVA), despite virus RNA still being detected on qPCR. c Red light demonstrated the maximal inactivation effectiveness, with significant difference in all scenarios (p < 0.001, one-way ANOVA), despite virus RNA still being detected on qPCR. Left panels: Black dots represent qPCR results in IU/mL in the control group whereas qPCR results in different MB concentration are shown in blue (1uM), red (05.uM), green (0.1uM), and white (0.01 uM). (white). Right panels: Black dots represent cluster count results in the control group whereas cluster count results in different MB concentration are shown in blue (1uM), red (05.uM), green (0.1uM), and white (0.01 uM). Centre line represents mean and bounds of box are standard deviation. Centre line represents mean and bounds of box are standard deviation. d Representative picture of infectivity loss in treated perfusate samples. Scale bar = 400 µm

Fig. 5

Pre-clinical large animal safety studies using EVLP/LbT treatments. a Schematic of a pre-clinical EVLP and lung transplantation model, designed to assess potential acute lung injury in donor lungs after LbT applied during EVLP (n = 4, each group): (1) Control (standard EVLP technique); (2) UVC (254 nm; 31 mW/cm²); (3) PDT, using 1μmol/L MB diluted in the perfusion solution associated with red light irradiation (660 nm; 20 mW/cm²). b–e Lung function parameters after left lung transplantation (N.S. after one-way ANOVA statistical analysis). f Graft inflammatory cytokine assessment in lung tissue after transplantation (N.S. after one-way ANOVA statistical analysis. Error bar indicate standard deviation)

Fig. 6

Pre-clinical large animal safety studies using EVLP/LbT treatments. a Lung injury score after transplantation, scale bar = 100 µm. b Cell death assessment (TUNEL) after transplantation, scale bar = 400um (N.S. after one-way ANOVA statistical analysis). MB: methylene blue; CIT: cold ischemia time; PDT: photodynamic therapy; Ultraviolet C (UVC) irradiation; EVLP: ex vivo lung perfusion; LbT: Light based therapy. Error bar indicates standard deviation

Fig. 7

Clonogenic cell assay performed in six-well plates, with clones produced by LL 24 ATCC ® CCL-151™ human fibroblasts. Steen was previously irradiated for 3 h while circulating in the mini-circuit and samples were taking hourly. a Cells were cultured for 12 days in a mixture of fresh Steen and media (control, upper images) and in a mixture of UVC-treated Steen and media (UVC, bottom images). No cytotoxic effect of the UVC was seen. b Survival fraction curves of LL 24 ATCC ® CCL-151™ human fibroblasts (n = 3 replicates). The survival curves derived from clonogenic assays experiments and are not significantly different, when comparing untreated controls with cells plated with irradiated Steen solution, after one-way ANOVA statistical analysis