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.     

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 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.     

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.     

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.     

Development of a recombinant antigen‐based ELISA for the sero‐detection of porcine lymphotropic herpesviruses

Abstract: Background: Xenotransplantation using pigs as donor species carries a risk for the activation of latent porcine herpesviruses and potential transmission to the human recipient. The porcine lymphotropic herpesviruses (PLHV‐1, ‐2, ‐3) are widespread in domestic pigs and closely related to the human gammaherpesviruses, Epstein–Barr virus and Kaposi sarcoma herpesvirus, causing lymphoproliferative disorders. PLHV‐1 has been associated with a porcine post‐transplantation lymphoproliferative disorder (PTLD), affecting miniature swine after experimental transplantation. In human xenotransplantation, PLHV might be transferred to the transplant recipient and cause PTLD or related diseases. The elimination of PLHV from donor pigs is therefore necessary, and requires the availability of nucleic‐acid‐ and antibody‐based detection methods. Methods: The N‐ and C‐terminal parts (gB1 and gB2) of the glycoprotein B gene of PLHV‐1, ‐2 and ‐3 were cloned and expressed in Escherichia coli. Antisera were raised in mice. PLHV PCR was performed as published earlier. Results: An ELISA was developed, using recombinant glycoprotein B of PLHV‐1 as the antigen, and used for the analysis of groups of pigs, differing by age and origin. Seropositivity ranged from 38% (piglets) to 90% (gilts) and 100% (breeding sows, miniature pigs and pigs for slaughter). In comparison, PCR products of PLHV were found in the blood of 0 to 80% of the pig groups. Additionally, a group of 12 piglets was tested repeatedly after birth until the age of 156 days. A decline of antibodies was found during the first 3 weeks after birth, followed by a rise in most pigs during the weeks thereafter. PLHV PCR products in the blood were only observed later than 3 weeks after birth. Conclusion: Newborn pigs may be passively protected by maternal antibodies against PLHV infection during the first 3 weeks post partum. The rise of antibody titers thereafter and the appearance of PLHV sequences in the blood possibly indicates de novo infection by contact to the infected mother sow. The PLHV–ELISA may aid in breeding PLHV‐free pigs.     

Examining the potential for porcine‐derived islet cells to harbour viral pathogens

With an onus on safety in the potential use of porcine islet cells as a treatment for diabetes, the use of animals lacking exogenous pathogens is clearly important and multilevel screening strategies have been presented on testing animals and the product. In this study, we wished to investigate whether islet cells indeed harboured the same viral pathogens of concern in the source animal. PMBC and islet cells from both adult and neonatal source animals were directly compared and tested for PCMV, PLHV, PCV2, PPV and HEV using both molecular and serological assays. Adult PBMC were found positive for all viruses with the exception of PCV2 and HEV. Neonatal PBMC were only found positive for PCMV and HEV. All animals were found negative for HEV antibodies. Interestingly, islet cells were negative for all viruses tested regardless of status in the animal‐derived PBMC. Given that other laboratories have demonstrated the lack of virus detection during the culture of islets, this study also demonstrates that the hygiene status of the herd may not reflect the status of the product. This is important for establishing guidelines for any risk evaluation and mitigation process utilised during product manufacture.     

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.     

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.     

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.     

Porcine Endogenous Retroviral Nucleic Acid in Peripheral Tissues Is Associated with Migration of Porcine Cells Post Islet Transplant

Porcine islets represent an alternative source of insulin-producing tissue, however, porcine endogenous retrovirus (PERV) remains a concern. In this study, SCID mice were transplanted with nonencapsulated (non-EC), microencapsulated (EC) or macroencapsulated (in a TheraCyte trade mark device) neonatal porcine islets (NPIs), and peripheral tissues were screened for presence of viral DNA and mRNA. To understand the role of an intact immune system in PERV incidence, mice with established NPI grafts were reconstituted with splenocytes. Peripheral tissues were screened for PERV and porcine DNA using PCR. Tissues with positive DNA were analyzed for PERV mRNA using RT-PCR. No significant difference was observed between non-EC and EC transplants regarding presence of PERV or porcine-specific DNA or mRNA. In reconstituted animals, little PERV or porcine DNA, and no PERV mRNA was detected. No PERV or porcine-specific DNA was observed in animals implanted with a TheraCyte trade mark device. In conclusion, an intact immune system significantly lowered the presence of PERV. Microencapsulation of islets did not alter PERV presence, however, macroencapsulation in the TheraCyte device did. Lower PERV incidence coincided with lower porcine DNA in peripheral tissues, linking the presence of PERV to migration of porcine cells.     

Engraftment of Adult Porcine Islet Xenografts in Diabetic Nonhuman Primates Through Targeting of Costimulation Pathways

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.     

Intraperitoneal alginate-encapsulated neonatal porcine islets in a placebo-controlled study with 16 diabetic cynomolgus primates

BACKGROUND: A nonhuman primate model of diabetes is valuable for assessing porcine pancreatic islet transplants that might have clinical benefits in humans. METHODS: Neonatal porcine islets, microencapsulated in alginate-polyornithine-alginate, were injected intraperitoneally (10,000 IEQs/kg islets) into eight adult male cynomolgus monkeys rendered diabetic with streptozotocin. Eight diabetic controls were given an equivalent dose of empty placebo capsules. All subjects received a repeat transplant 3 months after the first. RESULTS: The transplant was well tolerated and no adverse or hypoglycemic events occurred. There were two deaths from nontransplant treatment or diabetic complications unrelated to the transplants. After transplantation, the average insulin dose was reduced in the islet-treated group and increased in the control group. At 12 weeks after the first transplant there was a mean 36% (95% CI: 6% to 65%, P = .02) drop in daily insulin dose compared with the control group. After 24 weeks the difference increased to a mean of 43% (95% CI: 12% to 75%, P = .01) without significant differences in blood glucose values between the two groups. Individual responses after islet transplant varied and one monkey was weaned off insulin by 36 weeks. At terminal autopsy, organs appeared normal and there was no visible peritoneal reaction. No animal had polymerase chain reaction (PCR)-amplified signals of porcine endogenous retrovirus or exogenous virus infections in blood or tissues. CONCLUSION: Repeated intraperitoneal transplantation of microencapsulated neonatal porcine islets is a safe procedure in diabetic primates. It was shown to result in a significant reduction in insulin dose requirement in the majority of animals studied, whereas insulin requirement increased in controls.     

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.     

Accelerated functional maturation of isolated neonatal porcine cell clusters: in vitro and in vivo results in NOD mice

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/ differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the beta-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from < 3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in beta-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional beta-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.     

Molecular interactions between porcine and human gammaherpesviruses: implications for xenografts?

BACKGROUND: Reactivation of latent herpesviruses is an important cause of morbidity and mortality in human transplantation. This issue might be further complicated in the case of xenotransplantation. Zoonotic viruses could reactivate and replicate in the transplanted tissue, and interactions with homologous human viruses could take place. Since the pig is a favoured animal as donor of organs for human transplants, we analysed the possibility of interactions between porcine and human herpesviruses. Porcine lymphotropic herpesvirus 1 (PLHV-1) is a gammaherpesvirus homologous to Epstein-Barr virus (EBV) and to human herpesvirus 8 (HHV-8), is highly prevalent in pigs and is associated to lymphoproliferative disease in immunosuppressed and transplanted miniature swine. METHODS: The main viral transactivators of PLHV-1, ORF50, ORF57, ORFA6/BZLF1(h), were cloned and tested for their transactivating ability on several EBV and HHV-8 promoters using reporter assays. Also the effects of HHV-8 ORF50, ORF57 and ORFK8 and EBV BRLF1/ R-transactivator (Rta) and BZLF1/ Z-transactivator (Zta) on PLHV-1 lytic promoters were analysed. RESULTS: Porcine lymphotropic herpesvirus 1 ORF50 upregulated all HHV-8 promoters and PLHV-1 ORFA6/BZLF1(h) transactivated EBV promoters. Furthermore, transfection of PLHV-1 ORF50 into BC-3 cells, latently infected with HHV-8, resulted in HHV-8 reactivation. Likewise, HHV-8 ORF50 and EBV BRLF1/Rta had a strong transactivating effect on PLHV-1 promoters. Also EBV BZLF1/Zta and HHV-8 ORF57 induced PLHV-1 transactivation, but at lower levels. CONCLUSION: The results suggest that reciprocal molecular interactions between human and porcine herpesviruses might occur in vivo, and support the hypothesis that PLHV-1 might have pathogenic relevance in the course of xenotransplantation.