AN69 hollow fiber membrane will reduce but not abolish the risk of transmission of porcine endogenous retroviruses

As the risk of porcine endogenous retrovirus (PERV) infection is a major obstacle to the xenotransplantation of porcine tissue, we investigated whether an AN69 hollow fibre membrane, used for islets of Langerhans transplantation, could prevent the transfer of PERVs and thus reduce the risk of PERV infection. PK15 cells were used as a PERV source. A specific and highly sensitive RCR was used for detection of a PERV provirus DNA (gag region) and a porcine mtDNA. Human U293 cells were incubated in vitro with encapsulated PK15 cells, concentrated encapsulated PK15 supernatant, or concentrated PK15 supernatant as a control. CD1 mice were implanted in vivo with encapsulated PK15 cells or injected with PK15 supernatant. We found no infection in human cells incubated with either encapsulated PK15 supernatant or in 10 out of 11 samples after coincubation with encapsulated PK15 cells. Infection of human cells was, however, detected in 1 out of 11 samples after coincubation with encapsulated PK15 cells. The presence of PERV provirus DNA and porcine mtDNA was detected in all the investigated tissues of the mice injected with PK15 supematant and in various tissues of the mice implanted with encapsulated PK15 cells. Four weeks after the last injection of PK15 supernatant or a fiber explantation, no mouse showed any presence of PERV provirus DNA or porcine mtDNA. Our results demonstrate that AN69 hollow fiber membrane will reduce but not abolish the risk of PERV infection. Because the real risk of PERV infection still remains unknown, it is necessary to investigate further the real protection that could be provided by hollow fibers to ensure the safety of clinical xenotransplantation.     

Porcine endogenous retrovirus encodes xenoantigens involved in porcine cellular xenograft rejection by mice

BACKGROUND: Identification of the antigens that stimulate transplant rejection can help develop graft-specific antirejection strategies. The xenoantigens recognized during rejection of porcine cellular xenografts have not been clearly defined, but it has been assumed that major histocompatibility complex (MHC) xenoantigens are involved. METHODS: The role of porcine endogenous retrovirus (PERV) as a source of xenoantigens was examined. The authors used morphometry to compare the kinetics of swine leukocyte antigen (SLA) pig thyroid xenograft rejection in control mice and mice immunized with PERV PK15 cells (porcine kidney epithelial cells), PERV SLA pig peripheral blood lymphocytes (PBL), PERV virions purified from PK15 cells, and PERV or PERV A pseudotypes produced from infected human 293 cells. The tempo of rejection for cellular xenografts of PERV A pseudotype-producing human 293 cells, uninfected human 293 cells, and PK15 cells in PERV-preimmunized and control mice was also compared. RESULTS: Mice immunized with PK15 cells rejected pig thyroid xenografts significantly faster at day 5 than control mice and mice immunized with pig PBL. This correlated with the amount of PERV RNA and virions produced, but not with the amount of SLA class I MHC expressed by PK15 cells. Immunization of mice with PERV virions purified from porcine PK15 cells and with PERV virions or PERV A pseudotypes produced by human 293 cells also induced accelerated xenograft rejection by 5 days. Accelerated rejection induced by virus pretreatment was CD4 T-cell dependent and restricted to PERV-expressing cellular xenografts of porcine or nonporcine origin. CONCLUSIONS: PERV acts as a significant source of xenoantigens that target porcine cellular xenografts for rejection.     

Evidence of Absence of Porcine Endogenous Retrovirus (PERV) Infection in Patients Treated with a Bioartificial Liver Support System

Porcine endogenous retrovirus (PERV) genomes are present in all pig cells. In this retrospective study, we assessed PERV infectivity in 28 patients treated with an extracorporeal bioartificial liver (HepatAssist system) that includes a membrane device containing porcine hepatocytes. All patients tested negative for PERV using polymerase chain reaction analysis of peripheral blood mononuclear cells (PBMC) collected up to 5 years after treatment. In vitro results showed that the membrane decreased the risk of PERV transmission by a factor of 105, and porcine hepatocytes did not produce infectious PERV in co-cultures with human cell line 293. Our results do not support the presence of PERV infection in patients treated with this porcine hepatocyte-based bioartificial liver.     

The SlideReactor--a simple hollow fiber based bioreactor suitable for light microscopy

Most bioartificial liver support systems are based on hollow fiber capillaries within modified dialysis cartridges or more sophisticated bioreactor constructions. Due to their design microscopic follow-up of reorganization and growth of tissue between the hollow fibers is not possible. The SlideReactor is a simple hollow fiber based bioreactor construction suitable for light microscopy and time-lapse video observation. The SlideReactor offers a cell compartment separated from a medium inflow and outflow compartment. Cell compartment access ports enable easy filling of the cell compartment with cell suspension, as well as fixation of the tissue. For more complex procedures or full access to all the cells, the bioreactor can be opened easily by cutting the silicone seal with a scalpel. Due to its simple design and the utilization of standard materials, it could serve as a suitable, cost-efficient tool to evaluate the behavior of cells cultured between hollow fiber capillaries. The paper describes the production process: similar to open source projects in software engineering, we would like to propose the concept as an open platform to anyone interested in hollow fiber based cell culture.     

APOBEC3 proteins and porcine endogenous retroviruses

Xenotransplantation of porcine cells, tissues, and organs offers a solution to overcome the shortage of human donor materials. In addition to the immunological and physiological barriers, the existence of numerous porcine microorganisms including viruses poses a risk for xenozoonosis. Three classes of functional gamma-type porcine endogenous retroviruses (PERV) have been identified, whereby functional polytropic PERV-A and PERV-B infect human embryonic kidney (HEK 293) and other cell lines in vitro. In the course of risk assessment for xenotransplantation the capacity of human cells to counteract PERV infections should be analyzed. Primates and other mammals display different means of protection against viral infections. APOBEC3 proteins which are cytidine deaminases and a part of the intrinsic immunity mediate potent activity against a wide range of retroviruses including murine leukemia viruses (MLV). As PERV and MLV belong to the same genus, we raised the question as to whether PERV is affected by APOBEC3 proteins. Initial data indicate that human and porcine cytidine deaminases inhibit PERV replication, thereby possibly reducing the risk for infection of human cells by PERV as a consequence of pig-to-human xenotransplantation.     

Evaluation of water and electrolyte transport of tubular epithelial cells under osmotic and hydraulic pressure for development of bioartificial tubules

Our aim was to develop bioartificial tubules using tubular epithelial cells and artificial membranes and evaluate the function of water and electrolyte transport by various tubular epithelial cells. The cells were cultivated onto extracellular matrix (ProNectin F) coating polycarbonate membrane. Water transport from the apical to the basolateral site of cells was examined using a modified Ussing chamber module. Water transport under colloidal osmotic pressure on the apical site and hydraulic pressure on the basolateral site were higher in JTC-12, LLC-PK1 cells than in MDCK cells. Water transport under osmotic plus hydraulic pressure was highest in LLC-PK1 cells. We made bioartificial tubules using LLC-PK1 cells and polysulfone hollow fiber cartridges. Water and Na ion transport function was high, and BUN and creatinine passage was recognized in these bioartificial tubules. BUN and creatinine concentrations of reabsorption fluid in these bioartificial tubules were significantly lower than those concentrations of control media and of noncell attached polysulfone hollow fiber cartridges. Though LLC-PK1 cells were more preferable cells for the use of bioartificial tubules in terms of water and electrolyte transport, the passage of BUN and creatinine was not appropriate for clinical use. To select more preferable cells for bioartificial tubules which transport water and electrolytes and do not induce passage of uremic toxins is necessary.     

Natural antibodies prevent in vivo transmission of porcine islet-derived endogenous retrovirus to human cells

The discovery of porcine endogenous retroviruses (PERV) has raised concerns regarding the safety of porcine xenotransplantation. However, transmission of PERV had not been observed in humans exposed to porcine tissue. We examined whether PERV derived from porcine pancreatic islet cells could infect human cells in vivo and the role of natural antibodies in inhibiting PERV infection. In vivo infective potential of PERV was studied in SCID mice reconstituted with human peripheral blood leucocytes. Porcine islets were transplanted under the kidney capsule. PERV infection was determined by analyzing PERV gene expression in graft infiltrating lymphocytes (GIL) harvested 21 days posttransplantation. Mice were administered normal human serum prior to and 2 days posttransplantation to study their role in protection of human cells against PERV infection. PERV genes were expressed in all porcine tissues examined, including purified porcine islets. PERV expression was detected in GILs from three of five human-SCID mice. Administration of human serum blocked PERV infection in GILs in five of five human-SCID mice. These results indicate that PERV from porcine islets can infect human cells in vivo. Normal human serum blocks transmission of retrovirus in vivo, suggesting that natural xenoreactive antibodies can prevent PERV infection.     

Absence of PERV specific humoral immune response in baboons after transplantation of porcine cells or organs

Xenotransplantation of pig organs seems a promising way of overcoming the prevailing limitation on allotransplantation due to donor numbers. However, as porcine endogenous retroviruses (PERVs) can infect human cells in vitro, there is substantial concern regarding the risk of a PERV infection in xenogeneic transplant recipients. Cultured porcine endothelial cells, stimulated peripheral blood mononuclear cells, and pancreatic islet cells can release PERV infectious for human cells in vitro, but it is currently unknown whether PERV is released in vivo, whether these viral particles can infect the transplant recipient, and whether they are pathogenic. In a retrospective study 15 immunosuppressed baboons were tested for a specific immune response against PERV after transplantation of porcine endothelial cells, mononuclear blood cells, and lungs. Anti-PERV antibody expression was analyzed with peptide-based, enzyme-linked immunosorbent assays and highly sensitive Western Blot assays. This xenotransplantation study using nonhuman primates found no evidence of PERV specific humoral immune response. Our data suggest that no productive PERV infection and no continuous PERV release takes place in the nonhuman primates analyzed in this study.     

Development and validation of a Western immunoblot assay for detection of antibodies to porcine endogenous retrovirus

BACKGROUND: Reports that pig endogenous retrovirus (PERV) infects human cells in vitro have heightened the importance of molecular and serologic monitoring of xenograft recipients for evidence of infection with PERV. We report the development and validation of a PERV-specific Western immunoblot assay for the diagnostic testing of porcine xenografts recipients. This assay is based upon the serological cross-reactivity observed between PERV variants capable of infecting human cells in vitro and other mammalian C type retroviruses. METHODS AND RESULTS: Strong reactivity between PERV expressing embryonic pig kidney PK-15 cells and antisera raised against whole virus preparations of murine leukemia virus, gibbon ape leukemia virus (GALV), and simian sarcoma-associated virus was demonstrated by an immunofluorescence assay, suggesting specific antigenic cross-reactivity between this group of viruses and PERV. Western immunoblot analysis demonstrated that anti-GALV antisera reacted with three proteins in PK-15 cells having molecular masses of 30, 55, and 66 kDa. Antisera specific for the Gag proteins of either GALV or simian sarcoma-associated virus reacted with the 30-kDa (major) and 55-kDa (minor) proteins present in PK-15 cells and in PERV-infected 293 human kidney cells, likely representing reactivity to the processed and precursor forms of the PERV Gag protein, respectively. No reactivity was seen in uninfected 293 cells. Analysis of plasma samples from 200 United States blood donors and from 58 human immunodeficiency virus-1, 18 human immunodeficiency virus-2, 13 human T-cell lymphotrophic virus-I, 21 human T-cell lymphotrophic virus-II, and 15 cytomegalovirus infected controls were negative. CONCLUSIONS: As this assay is based on PERV antigen derived from infected human cells, it clearly has the capacity to detect a serologic response towards PERV variants that have zoonotic potential and will allow for the accurate determination of PERV-specific seroreactivity in porcine xenograft recipients.     

Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver

Di Nicuolo G, D’Alessandro A, Andria B, Scuderi V, Scognamiglio M, Tammaro A, Mancini A, Cozzolino S, Di Florio E, Bracco A, Calise F, Chamuleau RAFM. Long‐term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell–based Academic Medical Center bioartificial liver. Xenotransplantation 2010; 17: 431–439. © 2010 John Wiley & Sons A/S. Abstract: Background: Clinical use of porcine cell–based bioartificial liver (BAL) support in acute liver failure as bridging therapy for liver transplantation exposes the patient to the risk of transmission of porcine endogenous retroviruses (PERVs) to human. This risk may be enhanced when patients receive liver transplant and are subsequently immunosuppressed. As further follow‐up of previously reported patients (Di Nicuolo et al. 2005), an assessment of PERV infection was made in the same patient population pharmacologically immunosuppressed for several years after BAL treatment and in healthcare workers (HCWs) involved in the clinical trial at that time. Methods: Plasma and peripheral blood mononuclear cells (PBMCs) from eight patients treated with the Academic Medical Center‐BAL (AMC‐BAL), who survived to transplant, and 13 HCWs, who were involved in the trial, were assessed to detect PERV infection. A novel quantitative real‐time polymerase chain reaction assay has been used. Results: Eight patients who received a liver transplant after AMC‐BAL treatment are still alive under long‐term pharmacological immunosuppression. The current clinical follow‐up ranges from 5.6 to 8.7 yr after BAL treatment. A new q‐real‐time PCR assay has been developed and validated to detect PERV infection. The limit of quantification of PERV DNA was ≥5 copies per 1 × 10⁵ PBMCs. The linear dynamic range was from 5 × 10⁰ to 5 × 10⁶ copies. In both patients and HCWs, neither PERV DNA in PBMCs nor PERV RNA in plasma and PBMC samples have been found. Conclusion: Up to 8.7 yr after exposure to treatment with porcine liver cell–based BAL, no PERV infection has been found in long‐term immunosuppressed patients and in HCWs by a new highly sensitive and specific q‐real‐time PCR assay.     

Productive infection of primary human endothelial cells by pig endogenous retrovirus (PERV)

Abstract: The potential risk of viral transmission in the setting of xenotransplantation has gained major attention. Different porcine cell types have been shown to release retroviral particles, which are infectious for human cell lines in vitro. However, there are only a few data on whether PERV (pig endogenous retrovirus) is able to infect primary human cells. In this study we have analyzed endothelial cells, vascular fibroblasts, mesangial cells, mononuclear cells, hematopoetic stem cells and bone marrow stromal cells for PERV transmission. We now provide evidence for primary human endothelial cells, vascular fibroblasts, and mesangial cells to be susceptible to PERV transmission. PERV infection was productive in endothelial cells and mesangial cells. Our data confirm and extend former reports concerning the PERV infection of human cells. The PERV infection of different primary human cells represents further significant evidence for a viral risk during xenotransplantation. In this context, special attention should be directed towards productive infection of human endothelial cells: in the setting of xenotransplantation this cell type will have close contact with porcine cells and PERV particles.     

Safety of xenotransplantation: screening the donor pig and the recipient

Introduction: Xenotransplantation using pig cells and tissues may be associated with the transmission of porcine microorganisms including bacteria, parasites, fungi and viruses to the human recipient and may result in zoonones. Porcine endogenous retroviruses (PERVs) represent a special risk since PERV‐A and PERV‐B are present in the genome of all pigs and infect human cells. PERV‐C is not present in all pigs and does not infect human cells. However, recombinants between PERV‐A and PERV‐C have been observed in normal pigs characterised by higher replication rates compared with PERV‐A, and they are also able to infect human cells (1). Methods: In the past years numerous assays based on the PCR technology have been developed to screen for the prevalence and expression of PERV and other porcine microorganisms in the donor pig (2). Whereas most microorganisms may be eliminated by designated pathogen‐free breeding, PERVs cannot be removed this way. In addition, assays have been developed to analyse the recipient for the transmission of PERV and other microorganisms, either using PCR methods or immunological assays to detect an antibody production as a result of infection (3). Results: Using these assays, no transmission of PERV as well as of other porcine microorganisms has been observed in first preclinical and clinical xenotransplantations or animal infection experiments. This was especially true for the first clinical transplantation of pig islet cells approved by the New Zealand government (4). Until now there is no susceptible animal model to study PERV transmission and transplantations of porcine cells or organs to non‐human primates as they are associated with limitations concerning the safety aspect, which do not allow transmitting the negative findings to humans (5). Different experimental approaches are under development to reduce the probability of PERV transmission, e.g. the generation of transgenic pigs expressing PERV‐specific siRNA inhibiting PERV expression by RNA interference (6), genotypic selection of pigs with a low prevalence and expression of PERV and neutralising antibodies against the envelope proteins inhibiting PERV infection (7). Conclusion: Investigations of the last years resulted in highly sensitive and specific methods to study PERV and other microorganisms in donor pigs and human recipients of xenotransplants. These methods showed absence of PERV transmission in all investigated cases, both in more than 200 human xenotransplant recipients, mostly recipients of cellular xenotransplants, as well as in non‐human primates and small animals. New technologies under development may further decrease the probability of transmission. References: 1. Denner J. Recombinant porcine endogenous retroviruses (PERV‐A/C): A new risk for xenotransplantation? Arch Virol 2008; 153: 1421–1426. 2. Kaulitz D, Mihica D, Dorna J, Costa MR, Petersen B, Niemann H, TÖnjes RR, Denner J. Development of sensitive methods for detection of porcine endogenous retrovirus‐C (PERV‐C) in the genome of pigs J Virol Methods 2011; 175(1): 60–65. 3. Denner, J. Infectious risk in xenotransplantation – what post‐transplant screening for the human recipient? Xenotransplantation 2011; 18(3): 151–157. 4. Wynyard S, Garkavenko O, Nathu D, Denner J, Elliott R. Microbiological safety of the first clinical pig islet xenotransplantation trial in New Zealand, submitted. 5. Mattiuzzo G, Takeuchi Y. Suboptimal porcine endogenous retrovirus infection in non‐human primate cells: implication for preclinical xenotransplantation. PLoS One 2010; 5(10): e13203. 6. Semaan M, Kaulitz D, Petersen B, Niemann H, Denner J. Long‐term effects of PERV‐specific RNA interference in transgenic pigs. Xenotransplantation 2012; 19(2): 112–21. 7. Kaulitz D, Fiebig U, Eschricht M, Wurzbacher C, Kurth R, Denner J. Generation of neutralising antibodies against porcine endogenous retroviruses (PERVs). Virology 2011; 411(1): 78–86.