Infectious disease risks in xenotransplantation

Hurdles exist to clinical xenotransplantation including potential infectious transmission from nonhuman species to xenograft recipients. In anticipation of clinical trials of xenotransplantation, the associated infectious risks have been investigated. Swine and immunocompromised humans share some potential pathogens. Swine herpesviruses including porcine cytomegalovirus (PCMV) and porcine lymphotropic herpesvirus (PLHV) are largely species‐specific and do not, generally, infect human cells. Human cellular receptors exist for porcine endogenous retrovirus (PERV), which infects certain human‐derived cell lines in vitro. PERV‐inactivated pigs have been produced recently. Human infection due to PERV has not been described. A screening paradigm can be applied to exclude potential human pathogens from “designated pathogen free” breeding colonies. Various microbiological assays have been developed for screening and diagnosis including antibody‐based tests and qualitative and quantitative molecular assays for viruses. Additional assays may be required to diagnose pig‐specific organisms in human xenograft recipients. Significant progress has been made in the evaluation of the potential infectious risks of clinical xenotransplantation. Infectious risk would be amplified by intensive immunosuppression. The available data suggest that risks of xenotransplant‐associated recipient infection are manageable and that clinical trials can be performed safely. Possible infectious risks of xenotransplantation to the community at large are undefined but merit consideration.     

The 2017 IXA Presidential Lecture: Recent developments in xenotransplantation

The last few years have seen significant progress in xenotransplantation. Porcine xenograft survival in preclinical models continues to improve, accompanied by the adjustment of immunosuppression to more clinically realistic levels. The rapid uptake of CRISPR/Cas9 technology has accelerated the generation of new knock‐in and knockout pigs, including animals null for the endogenous retrovirus PERV. This brief review presents a personal view of recent developments in the field.     

Retroviral Restriction Factors and Infectious Risk in Xenotransplantation

The clinical application of xenotransplantation poses immunologic, ethical, and microbiologic challenges. Significant progress has been made in the investigation of each of these areas. Among concerns regarding infectious risks for human xenograft recipients is the identification in swine of infectious agents including porcine endogenous retroviruses (PERV) that are capable of replication in some human cell lines. PERV replication has, however, been difficult to demonstrate in primate‐derived cell lines and in preclinical studies of non‐human primates receiving porcine xenografts. Endogenous ‘retroviral restriction factors’ are intracellular proteins and components of the innate immune system that act at various steps in retroviral replication. Recent studies suggest that some of these factors may have applications in the management of endogenous retroviruses in xenotransplantation. The risks of PERV infection and the potential role of retroviral restriction factors in xenotransplantation are reviewed in detail.     

Infectious risk and retroviral restriction factors in xenotransplantation

The clinical application of xenotransplantation evokes immunological and microbiological as well as virological challenges. Porcine pathogens that do not show any symptoms in their natural host could exhibit a risk of fatal infections to humans. The presence of pig infectious agents including zoonotic and dissimilar agents should be reduced by specific pathogen free (spf) breeding of donor animals. However, the genetic information of porcine endogenous retroviruses (PERV) is integrated in the pig genome and can not be eradicated by spf breeding. The concerns about PERV for human xenograft recipients are based on data of in vitro replication of PERV in some human cell lines. So far, viral replication of PERV has been difficult to demonstrate in non‐human primate cell lines and in preclinical studies of non‐human primates receiving porcine xenografts, respectively. In this regard, natural and effective mechanisms of human and porcine cells counteracting productive infections caused by PERV are important to investigate. Intracellular proteins and components of the innate immune system including endogenous “antiretroviral restriction factors” act at various steps in retroviral replication. The cellular front is composed by several constitutively expressed genes which prevent or suppress retroviral infections. Some of these factors such as members of the tripartite motif (TRIM) and the apolipoprotein B mRNA‐editing polypeptide (APOBEC) families as well as tetherin and zinc‐finger antiviral protein (ZAP) could be useful in the management of PERV in xenotransplantation. The risks of infection and the potential role of antiretroviral restriction factors in xenotransplantation are presented in detail.     

Microbiological safety of xenotransplantation compared with allotransplantation

Transmission of viral, bacterial, parasitic, and fungal infections via organ allografts is uncommon but may be associated with life‐threatening disease. Internationally, programs for screening of human organ donors for infectious risk are non‐uniform and vary with national standards and the availability of screening assays. Further, the failure to recognize and/or to report transmission events limits the utility of available data regarding the incidence of allograft‐associated disease transmission. Advances in xenotransplantation biology have allowed some limited clinical trials with the prospect for increased opportunities for clinical xenotransplantation. As with human allotransplantation, the examination of infectious risk has been a central theme in these studies. Significant advances have been made in the breeding and screening of swine for preclinical studies including the identification of novel, potential human pathogens derived from source animals. Thus far, “expected” xenograft‐derived pathogens such as porcine cytomegalovirus (PCMV) have become activated in immunosuppressed primates but have not resulted in systemic infection outside the xenograft. PCMV has been bred out of swine herds by early weaning strategies. Conversely, host pathogens such as primate‐derived cytomegalovirus (CMV) have become activated and have produced serious infectious complications. These infections are preventable using antiviral prophylaxis. Xenogeneic tissues appear to be relatively resistant to infection by common human pathogens such as HIV, HTLV and the hepatitis viruses. Concerns regarding the potential activation of latent porcine retroviruses from xenograft tissues have resulted in the development of novel assays for xenotropic porcine endogenous retrovirus (PERV). PERV transmission to primate xenograft recipients or to human cells in in vivo models has not been detected. Multiple intrinsic cellular mechanisms appear to be active in the prevention of infection of human cells by PERV. Further, PERV appears to be susceptible to available antiretroviral agents. Thus, while the absolute risk for such infections remains unknown in the absence of human studies with prolonged graft survival in immunosuppressed xenograft recipients, the risk of transmission to human recipients appears limited. Some general principles have been developed to guide clinical trials: Outcomes of xenotransplantation trials, including any infectious disease transmissions, should be reported in the scientific literature and to appropriate public health authorities. Surveillance programs should be developed to detect known infectious agents as well as previously unknown or unexpected pathogens in the absence of recognizable clinical syndromes. Standardization of procedures and validation by expert and/or reference laboratories are needed for microbiological assays. Such validation may require international collaboration. Repositories of samples from source animals and from recipients prior to, and following xenograft transplantation are essential to the investigation of possible infectious disease events. Infection is common in allograft recipients. Thus, in advance of clinical trials, policies and procedures should be developed to guide the evaluation of any infectious syndromes that may develop. (e.g. fever of unknown origin [FUO], leukocytosis, leukopenia, graft dysfunction, pneumonia, hepatitis, abscess formation) in xenograft recipients. Based on preclinical experience these procedures will include: (i) Exclusion of infectious syndromes commonly associated with allotransplantation (e.g. CMV, bacterial pneumonia); (ii) Evaluation of PERV infection by serologic and NAT testing; (iii) Assessment of other recipients of xenografts derived from the same herd or source of swine; and (iv) Evaluation of social contacts of the recipient. Consideration of investigation of xenograft recipients for unknown pathogens may require application of advanced research technologies, possibly including use of broad‐range molecular probes, microarrays or high throughput pyrosequencing. References: 1. Meije Y, TÖnjes RR, Fishman JA. Retroviral restriction factors and infectious risk in xenotransplantation. Amer J Transplant 2010; 10: 1511–1516. 2. Fishman JA, Scobie L, Takeuchi Y. Xenotransplantation‐associated infectious risk: A WHO consultation. Xenotransplantation 2012; 19: 72–81.     

Generation and testing of a highly specific anti-serum directed against porcine endogenous retrovirus nucleocapsid

Advances in xenotransplantation offer chances to alleviate the shortage of human donor organs. The discovery that pig endogenous retroviruses (PERV) can infect human cells in vitro has stimulated the discussion on infectious risk in xenotransplantation. A molecular and immunologic monitoring of xenograft recipients and of donor animals for putative infection with PERV and other microorganisms is inevitable. In this report, we describe the generation and testing of a highly specific anti-serum directed against the PERV nucleocapsid protein. The Gag amino acid (aa) sequence of PERV class B was used to define immunogenic domains by computer analysis. A peptide corresponding to the C-terminal 19 aa of the 10 kDa (p10) nucleocapsid (NC) portion of the Gag polyprotein was used to immunize rabbits. The generated serum was tested using recombinant PERV Gag protein expressed in insect cells, purified PERV virus particles and human 293 cells transfected or infected with PERV, respectively. Test methods included Western blotting, indirect immunofluorescence, immunoperoxidase assay and ELISA. The PERV anti-serum provides a tool that is instrumental for detection of a potential agent of zoonosis. It can be used for screening of donor animals and xenograft recipients in the course of xenotransplantation procedures.     

Xenotransplantation: Infectious Risk Revisited

Xenotransplantation is a possible solution for the shortage of tissues for human transplantation. Multiple hurdles exist to clinical xenotransplantation, including immunologic barriers, metabolic differences between pigs--the source species most commonly considered--and humans, and ethical concerns. Since clinical trials were first proposed almost 10 years ago, the degree of risk for infection transmitted from the xenograft donor to the recipient has been extensively investigated. A number of potential viral pathogens have been identified including porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV), and porcine lymphotropic herpesvirus (PLHV). Sensitive diagnostic assays have been developed for each virus. Human-tropic PERV are exogenous recombinants between PERV-A and PERV-C sequences and are present in only a subset of swine. Porcine cytomegalovirus can be excluded from herds of source animals by early weaning of piglets. In contrast, the risks associated with PLHV remain undefined. Microbiologic studies and assays for potential xenogeneic pathogens have furthered understanding of risks associated with xenotransplantation. Thus far, clinical xenotransplantation of pig tissues has not resulted in transmission of viral infection to humans; significant risks for disease transmission from swine to humans have not been confirmed. If immunologic hurdles can be overcome, it is reasonable to initiate carefully monitored clinical trials.     

Xenotransplantation literature update,November/December 2019

The ever-increasing disparity between the lack of organ donors and patients on the transplant waiting list is increasing worldwide. For the past several decades xenotransplantation has led the way to correct this deficit and remains clearly the only feasible option to provide a means to meet the demand for patients in need of an organ transplant. Xenotransplantation’s ability to provide a specifically designed unlimited supply of organs, suited to treat the various needs for transplant organs and cells, has recently been championed by successful pre-clinical trials that have run long-term in non-human primate studies. In this review we show how these improvements have come about due to long-term dedicated research and recent advances in biomedical engineering technology, such as genome editing tools including zinc finger nucleases, TALEN, and CRISPER/Cas9 which have paved the way for significant breakthroughs in improving xenograft outcomes through genetic modifications to the donor source pig. Other novel approaches include the development of decellularized porcine tissue, such as corneas which can now be transplanted into patients with the minimal need for immunosuppression or other side effects. Further genetic variants of the porcine genome are also now being optimized to abrogate rejection. The emergence of new modalities such as; mesenchymal stem cells, donor thymic vascularization, in vivo bioreactors, chemokine and cytokine therapies have come to show improvements in xenograft outcomes. Furthermore, new studies confirm the safety status of using porcine xenografts, verifying that with current technologies and approaches, the issue of PERV transmission is a moot point. These breakthroughs and technological advancements push the reality of xenotransplantation one step closer to the clinic.     

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.     

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.     

Porcine endogenous retrovirus transmission characteristics from a designated pathogen-free herd

Previously, a strategy for monitoring pigs intended for cell transplantation was developed and successfully applied to several representative herds in New Zealand. A better understanding of porcine viruses' epidemiology in New Zealand has been achieved, and, as a result, a designated pathogen-free (DPF) herd has been chosen as a good candidate for xenotransplantation. This herd is free of all infectious agents relevant to xenotransplantation. The presented study of pig endogenous retrovirus (PERV) transmission with cocultures in vitro has shown no evidence of PERV transmission from DPF pig tissue. Additionally, in PERV-C-positive DPF donor pigs tested, a specific locus for PERV-C present in miniature swine possibly associated with the transmission of PERV was absent. The data on PERV transmission allowed classifying the DPF potential donors as "null" or noninfectious pigs.     

Analysis of potential porcine endogenous retrovirus (PERV) transmission in a whole-organ xenotransplantation model without interfering microchimerism

The question whether porcine xenografts can lead to porcine endogenous retrovirus (PERV) infection of recipients is critical for the evaluation of the safety of pig-to-man xenotransplantation. Unfortunately, polymerase chain reaction (PCR)-based analysis of potential PERV infections in nonhuman-primate whole-organ xenotransplantation models is hampered by false positive results due to chimeric porcine cells. To avoid the inherent analytical problem of xenomicrochimerism, we developed a non-life-supporting pig-to-primate kidney xenotransplantation model: porcine kidneys were transplanted, whereas the functioning recipient kidneys remained in situ. Subsequent to rejection (after 2 hours to 15 days), xenografts were removed, and recipients remained alive for up to 287 days. Immunosuppressive therapy based on cyclophosphamide, cyclosporine, and steroids was maintained for 28 days after transplantation. Using appropriate PCR assays, xenochimerism was found in tissue samples and partly even in peripheral blood leukocytes (PBLs) while the porcine kidneys were in situ. After graft removal, xenochimerism was no longer detectable, thus allowing analysis for possible PERV transmission.     

No transmission of porcine endogenous retrovirus after transplantation of adult porcine islets into diabetic nude mice and immunosuppressed rats

BACKGROUND: The aim of this study was to investigate whether transmission of porcine endogenous retrovirus (PERV) occurs in a model of diabetes reversal by the xenotransplantation of adult porcine islets (APIs) into immunoincompetent diabetic rodents. METHODS: Black-6 nu/nu mice and Lewis rats were immunosuppressed with cyclosporin A (CsA) and FTY 720, and rendered diabetic with streptozotocin. Purified APIs were transplanted into the renal subcapsular space; 5,000 islet equivalents (IEQs) were used in the nude mice (n = 4) and 40,000 IEQs in the rats (n = 4). The nude mice were sacrificed at 75 days after transplantation. In order to confirm chronic xenograft function, the graft-bearing kidney was removed prior to sacrifice. The rats were followed until xenograft rejection, at which time they were sacrificed. Immediately after sacrifice, tissue samples (liver, spleen, and small intestine) were taken for analysis. Quantitative polymerase chain reaction (PCR) was used to assess evidence of PERV transmission, and porcine cell chimerism. RESULTS: All animals became normoglycemic within 48 h of transplantation. The nude mice remained normoglycemic during the 75-day study period, with removal of the graft-bearing kidney resulting in prompt hyperglycemia. The rats remained normoglycemic until xenograft rejection, which occurred at 66 +/- 28 days. Despite the evidence of porcine cell microchimerism in recipients, real-time PCR detected no evidence of PERV transmission in any of the tissue specimens tested. CONCLUSIONS: There was no evidence of PERV transmission following transplantation of pig islets into diabetic nude mice and immunosuppressed rats.     

Clinical Xenotransplantation: Pigs Might Fly?

Xenotransplantation has the potential to deliver an unlimited supply of organs for transplantation. However, this promise has yet to translate into clinical application, despite substantial research efforts in the last decade. Although increasing numbers of studies are being performed in relevant pre-clinical (pig-to-primate) transplantation models, so far these have highlighted the apparent elusiveness of long-term xenograft survival. Humoral rejection remains the main obstacle to success, but control of T cell-mediated rejection will be a problem in the future and there are major concerns about the possible transmission of porcine endogenous retroviruses (PERV) and other infectious agents. This article reviews recent advances in the understanding of acute vascular rejection (AVR), acute T cell-mediated rejection and PERV transmission and highlights some of the strategies that may prove successful in overcoming these problems. Although progress has been slow, the promise of an inexhaustible supply of organs is sufficient reason to continue research in these areas. Assuming the specific problem of AVR can be ameliorated by one of a number of strategies currently under investigation, there are grounds to believe that xenotransplantation will become a clinical reality. Pig xenografts, currently grounded, might eventually fly!     

Virus safety in xenotransplantation: first exploratory in vivo studies in small laboratory animals and non-human primates

For xenotransplantation, the transplantation of animal cells, tissues and organs into human recipients, to date, pigs are favored as potential donors. Beside ethical, immunological, physiological and technical problems, the microbiological safety of the xenograft has to be guaranteed. It will be possible to eliminate all of the known porcine microorgansims in the nearby future by vaccinating or specified pathogen-free breeding. Thus, the main risk will come from the porcine endogenous retroviruses (PERVs) which are present in the pig genome as proviruses of different subtypes. PERVs will therefore be transmitted, with the xenograft, to the human recipient. PERVs can infect numerous different types of human primary cells and cell lines in vitro and were shown to adapt to these cells by serial passaging on uninfected cells. Furthermore, PERVs have high homology to other retroviruses, such as feline leukemia virus (FeLV) or murine leukemia virus (MuLV), which are known to induce tumors or immunodeficiencies in the infected host. To evaluate the potential risk of a trans-species transmission of PERV in vivo, naive and immunosuppressed rats, guinea pigs and minks were inoculated with PERV and screened over a period of 3 months for an antibody reaction against PERV proteins or for the integration of proviral DNA into the genomic DNA of the host's cells. Furthermore, we inoculated three different species of non-human primates, rhesus monkey (Macaca mulatta), pig-tailed monkey (Macaca nemestrina) and baboon (Papio hamadryas) with high titers of a human-adapted PERV. To simulate a situation in xenotransplantation, the animals received a daily triple immunosuppression using cyclosporine A, methylprednisolone and RAD, a rapamycin derivative, presently under development by Novartis. None of the small laboratory animals or the non-human primates showed production of antibodies against PERV or evidence of integration of proviral DNA in blood cells or cells of several organs, 3 months after virus inoculation, despite the observation that cells of the animals used in the experiment were infectible in vitro. This apparent difference in the outcome of the in vitro and the in vivo data might be explained by an efficient elimination of the virus by the innate or adaptive immunity of the animals.     

Porcine pathogens and xenotransplantation: PERV and beyond!

Concerns regarding the transmission of potentially zoonotic porcine viruses via a xenotransplant have prompted a significant number of studies on methods to eliminate or prevent expression and transmission of these viruses. The main focus of these studies, to date, has been the porcine endogenous retrovirus (PERV); PERV is a genetically acquired element and present in the genome of all swine. This situation is problematic as it cannot simply be eliminated from swine by using methods currently employed to exclude exogenous pathogens in barrier facilities. As such, alternative strategies have been sought to circumvent the potential risk of PERV expression and transmission via a xenotransplant, however, there are other existing and emerging pathogens of concern that should be addressed when using this novel technology in vivo. Zoonotic porcine viruses have been identified that require specific diagnostic methods to confirm their absence. Animal husbandry and the exclusion of pathogens from SPF herds for use in xenotransplantation have been widely discussed and a number of organizations have issued guidelines on the screening for infectious agents. Although these recommendations on monitoring protocol and the identification of adventitious agents are clear, there is no comprehensive list of pathogens to be excluded from these animals that can be applied to all centres carrying out xenotransplantation. Currently, SPF animals used for research purposes are monitored for specific pathogens as defined by local guidelines, and may not be tested for all pathogens relevant to xenotransplantation. As recent data has indicated the potential for certain porcine pathogens to cross the species barrier, it is clear that xenotransplantation is a unique situation which may require us to address a more comprehensive panel of microorganisms than is currently recommended for SPF animals. This presentation will discuss data on the presence of pathogens in pigs, other than PERV, that may cause concern during the clinical application of xenotransplantation and the issues regarding the potential transfer of new zoonotic microorganisms.