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.     

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.     

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.     

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.     

Pig endogenous retroviruses and xenotransplantation

Xenotransplantation of porcine organs might provide an unlimited source of donor organs to treat endstage organ failure diseases in humans. However, pigs harbour retroviruses with unknown pathogenic potential as an integral part of their genome. While until recently the risk of interspecies transmission of these porcine endogenous retroviruses (PERV) during xenotransplantation has been thought to be negligible, several reports on infection of human cells in vitro and spread of PERV from transplanted porcine islets in murine model systems have somewhat challenged this view. Here, we compile available data on PERV biology and diagnostics, and discuss the significance of the results with regard to the safety of clinical xenotransplantation.     

Herpesvirus infections in xenotransplantation: pathogenesis and approaches

Infectious risk remains an important consideration in the clinical application of xenotransplantation. Vascularized xenografts create unique immunological niches in which bidirectional transmission of pathogens between donor and recipient may occur. Enhanced replication of many pathogens is stimulated by the immune responses induced by transplantation and by the immune suppression used to prevent graft rejection. Herpesviruses are the prototype viruses that are activated during immunosuppression. Quantitative diagnostic molecular assays have been developed for the known herpesviruses causing infection in pigs. Recent data suggest that some herpesviral infections, such as porcine cytomegalovirus, may be excluded from swine used as source animals by careful breeding, while others will require novel strategies for control. This review focuses on porcine and baboon herpesviruses in pig-to-non-human primate solid organ xenotransplantation including direct effects (tissue damage), indirect effects (coagulopathy, rejection), and possible approaches to these infections.     

Xenotransplantation-associated infectious risk: a WHO consultation

Xenotransplantation carries the potential risk of the transmission of infection with the cells or tissues of the graft. The degree of risk is unknown in the absence of clinical trials. The clinical application of xenotransplantation has important implications for infectious disease surveillance, both at the national and international levels. Preclinical data indicate that infectious disease events associated with clinical xenotransplantation from swine, should they occur, will be rare; data in human trials are limited but have demonstrated no transmission of porcine microorganisms including porcine endogenous retrovirus. Xenotransplantation will necessitate the development of surveillance programs to detect known infectious agents and, potentially, previously unknown or unexpected pathogens. The development of surveillance and safety programs for clinical trials in xenotransplantation is guided by a "Precautionary Principle," with the deployment of appropriate screening procedures and assays for source animals and xenograft recipients even in the absence of data suggesting infectious risk. All assays require training, standardization and validation, and sharing of laboratory methods and expertise to optimize the quality of the surveillance and diagnostic testing. Investigation of suspected xenogeneic infection events (xenosis, xenozoonosis) should be performed in collaboration with an expert data safety review panel and the appropriate public health and competent authorities. It should be considered an obligation of performance of xenotransplantation trials to report outcomes, including any infectious disease transmissions, in the scientific literature. 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. Concerns over any potential hazards associated with xenotransplantation may overshadow potential benefits. Careful microbiological screening of source animals used as xenotransplant donors may enhance the safety of transplantation beyond that of allotransplant procedures. Xenogeneic tissues may be relatively resistant to infection by some human pathogens. Moreover, xenotransplantation may be made available at the time when patients require organ replacement on a clinical basis. Insights gained in studies of the microbiology and immunology of xenotransplantation will benefit transplant recipients in the future. This document summarizes approaches to disease surveillance in individual recipients of nonhuman tissues.     

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.     

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.     

Microbiological safety of porcine islets: comparison with source pig

Abrahante JE, Martins K, Papas KK, Hering BJ, Schuurman H‐J, Murtaugh MP. Microbiological safety of porcine islets: comparison with source pig. Xenotransplantation 2011; 18: 88–93. © 2011 John Wiley & Sons A/S. Abstract: Background: Pig islet donors intended for clinical xenotransplantation for the treatment of diabetes must meet stringent conditions. Among others, viruses with the potential to cross the species barrier should be excluded from the herd: this list includes encephalomyocarditis virus (EMCV), hepatitis E virus (HEV), porcine cytomegalovirus (PCMV) and porcine γ‐lymphotropic herpesvirus (PLHV). As an islet product is isolated from the pancreas and then subjected to culture before implantation, the question is raised whether islets could be negative even if the animal itself is positive for a distinct pathogen. Methods: To answer this question, sensitive quantitative real‐time PCR assays were established for EMCV, HEV, PCMV and PLHV. Twelve adult animals from a high‐hygienic herd were then evaluated; testing tissues, where the virus is expected to reside in latent infection, testing islets immediately after isolation, and then isolated islets after a 7‐day culture. Results: None of the tissues tested positive for EMCV, HEV or PLHV. PCMV was observed in spleen tissue from six animals: three of these six animals were positive for isolated islets, and two of these three cases were also positive for islets after culture. Older animals in particular showed positivity, and within a given litter not all animals were PCMV positive. Conclusions: These data fit with spread through the herd by horizontal transmission, not in utero infection. PCMV has to be excluded from the herd to ensure that islets for transplantation are negative for PCMV.     

Is porcine endogenous retrovirus (PERV) transmission still relevant?

Xenotransplantation using porcine cells or organs may be associated with the risk of transmission of zoonotic microorganisms. Porcine endogenous retroviruses (PERVs) pose a potentially high risk because they are integrated into the genome of all pigs and PERV-A and PERV-B at least, which are present in all pigs, can infect human cells. However, PERV transmission could not be demonstrated in the first recipients of clinical xenotransplantation or after numerous experimental pig-to-non-human primate transplantations. In addition, inoculation of immunosuppressed small animals and non-human primates failed to result in demonstrable PERV infection. Nevertheless, strategies to reduce the possible danger of PERV transmission to humans, however low, could be of benefit for the large-scale clinical use of porcine xenotransplants. One strategy is to select pigs free of PERV-C, thereby preventing recombination with PERV-A. A second strategy involves the selection of animals that express only very low levels of PERV-A and PERV-B. To this end, sensitive and specific methods have been developed to allow the distribution and expression of PERV to be analyzed. A third strategy is to develop a vaccine capable of protecting against PERV transmission. Finally, a fourth strategy is based on the inhibition of PERV expression by RNA interference. Using PERV-specific short hairpin RNA (shRNA) and retroviral vectors, inhibition of PERV expression in primary pig cells was demonstrated and transgenic pigs were generated that show reduced PERV expression in all tissues analyzed. Intensive work is required to improve and to combine these strategies to further decrease the putative risk of PERV transmission following xenotransplantation.     

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.     

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.     

Methods for the exclusion of circoviruses and gammaherpesviruses from pigs

Abstract: The use of porcine tissues is being developed as a means to alleviate the shortage of allogeneic tissues and organs available for transplantation. To reduce the possibility of a microorganism of pigs being inadvertently transferred to the recipient of the xenograft, recommendations have been published on the microbiological specifications for organ source pigs. The porcine circoviruses (PCV1 and PCV2) and porcine lymphotropic herpesviruses (PLHV1 and PLHV2) are two infectious agents of pigs which are considered to be of significance for the microbiological safety of xenotransplantation. To ensure the exclusion of these microorganisms from animals destined for use under clinical conditions, reliable breeding methodologies are required. We investigated the efficiency of established derivation procedures for the removal of PCV and PLHV. In comparison with conventionally reared pigs, caesarian and barrier derived animals showed a markedly reduced prevalence of PCVs and PLHVs. Our results indicate that the derivation of animals free of both of these microorganisms is achievable and will enhance the microbiological safety of xenotransplantation.     

Early weaning of piglets fails to exclude porcine lymphotropic herpesvirus

BACKGROUND: Xenotransplantation using pigs as source species carries a risk for the activation of latent herpesviruses from the porcine donor and potential transmission to the recipient. In pig-to-baboon xenotransplantation, activation of porcine cytomegalovirus (PCMV) has been associated with xenograft injury and an increased incidence of consumptive coagulopathy and graft loss. Activation of porcine lymphotropic herpesvirus (PLHV)-1 was not observed in pig-to-baboon solid organ xenotransplantation, but was associated with a syndrome of post-transplantation lymphoproliferative disorder (PTLD) after allogeneic stem cell transplantation in pigs. MATERIAL and METHODS: Early weaning of piglets was used to try to reduce the viral burden of xenograft donors. This consisted of separating the piglets of a litter from the sow within the first 2 weeks after birth and raising them in isolation from the remaining herd. RESULTS: We have previously demonstrated that PCMV could be excluded from source animals by early weaning of piglets. However, early weaning failed to exclude PLHV-1 from source pigs. CONCLUSIONS: This disparity between PCMV and PLHV-1 reflects differing pathogenesis of infection of these herpesviruses. New approaches will be needed to exclude PLHV-1 from pig colonies.