Infectious risk in xenotransplantation – what post‐transplant screening for the human recipient?

Xenotransplantation may be associated with the transmission of pig microorganisms including viruses, bacteria, fungi, and parasites. As the recipient may be immunosuppressed, infection and pathologic consequences may be more pronounced compared to non-immunosuppressed individuals. Transmission of most microorganisms with exception of porcine endogenous retroviruses (PERV) may be prevented by screening the donor pig and qualified pathogen-free breeding. PERVs represent a special risk as they are present in the genome of all pigs and infect human cells in vitro. Until now, no PERV transmission was observed in experimental and clinical xenotransplantations as well as in numerous infection experiments. Nevertheless, several strategies have been developed to prevent PERV transmission.     

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.     

Xenotransplantation and Hepatitis E virus

Xenotransplantation using pig cells, tissues and organs may be associated with the transmission of porcine microorganisms to the human recipient. Some of these microorganisms may induce a zoonosis, that is an infectious disease induced by microorganisms transmitted from another species. With exception of the porcine endogenous retroviruses (PERVs), which are integrated in the genome of all pigs, the transmission of all other microorganisms can be prevented by specified or designated pathogen‐free (spf or dpf, respectively) production of the animals. However, it is becoming clear in the last years that the hepatitis E virus (HEV) is one of the viruses which are difficult to eliminate. It is important to note that there are differences between HEV of genotypes (gt) 1 and gt2 on one hand and HEV of gt3 and gt4 on the other. HEV gt1 and gt2 are human viruses, and they induce hepatitis and in the worst case fatal infections in pregnant women. In contrast, HEV gt3 and gt4 are viruses of pigs, and they may infect humans, induce commonly only mild diseases, if any, and are harmless for pregnant women. The goal of this review was to evaluate the risk posed by HEV gt3 and gt4 for xenotransplantation and to indicate ways of their elimination from pigs in order to prevent transmission to the human recipient.     

Sensitive and specific immunological detection methods for porcine endogenous retroviruses applicable to experimental and clinical xenotransplantation

Abstract: The use of organs from transgenic pigs for xenotransplantation may be associated with the risk of transmission of microorganisms, especially when the transgenic pigs express human proteins influencing complement activation. The porcine endogenous retroviruses (PERVs) are of particular concern as they can infect human cells in vitro. However, it is unknown whether PERVs can infect transplant recipients in vivo and, if so, whether they are pathogenic. It is therefore essential for experimental and clinical xenotransplantation procedures that specific and sensitive screening methods for PERVs are established. We developed Western blot and enzyme-linked immunosorbant assays (ELISA) based on purified PERVs produced by pig and human cells or recombinant viral protein and synthetic peptides corresponding to PERVs' transmembrane envelope protein, respectively. PERV-specific anti-sera generated against purified virus particles, purified viral proteins and synthetic peptides served as positive controls. Both assays were used for screening the sera of healthy blood donors, pregnant women, patients treated with pig tissues, and butchers with extensive contact to living porcine material to detect antibodies against PERV. None of the individuals showed an antibody pattern characteristic for retroviral infections. Some individuals had antibodies reactive against the major capsid protein p27, against smaller viral proteins of the group specific antigen (Gag) in Western blot assays, or against peptides in the ELISA, probably due to cross-reactivity. Here, we present specific and highly sensitive screening methods applicable for future xenotransplantation procedures, but using these methods we found no evidence of PERV-infection among humans potentially at risk.     

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.     

Review on porcine endogenous retrovirus detection assays—impact on quality and safety of xenotransplants

Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non‐sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell‐based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen‐free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV‐A/‐B/‐C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients.     

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.     

Can human viruses infect porcine xenografts?

Millard AL, Mueller NJ. Can human viruses infect porcine xenografts? Xenotransplantation 2010; 17: 6–10. © 2010 John Wiley & Sons A/S. Abstract: Xenotransplantation exposes the recipient to known and unknown pathogens of the donor pig (donor‐derived xenosis). A major effort has been undertaken to minimize the risk of transmission from the donor using specialized breeding techniques. With the exception of endogenous retroviruses and porcine lymphotropic herpesvirus, exclusion of known pathogens was successful and has eliminated a majority of donor pathogens. In the recipient, enhanced replication of many pathogens will be stimulated by the immune responses induced by transplantation and by the immune suppression used to prevent graft rejection. Infection of the graft may occur with unpredictable consequences due to the cross‐species situation. Infectivity may be decreased as entry or replication is altered by missing receptors or inability to use the cellular machinery. Replication of organisms in the xenograft and the inability of the human host to respond to human pathogens in the context of a xenograft infection due to immune suppression, or the presentation of such pathogens in the context of pig instead of human major histocompatibility complex (MHC) could impair control of such infections. Recent data suggest that some human herpesviruses infections, such as human cytomegalovirus, may infect porcine tissue and are associated with a pro‐inflammatory phenotype. This review focuses on human or recipient‐derived pathogens and their potential harmful role in xenograft infection.     

The lack of inhibition of porcine endogenous retrovirus by small interference RNA designed from the long terminal regions

Xenotransplantation from pigs may offer a potential solution to the organ shortage. However, there remains the risk of xenoinfection by porcine endogenous retroviruses (PERVs) that cannot be eliminated by breeding pigs under specified pathogen-free conditions. RNA interference is a new method to inhibit the expression of a specific gene. Here, we designed two siRNAs from the long terminal repeat of PERV. Our results showed that these siRNAs had no inhibitory effects. The possible reasons for this are an off-target effect or a problem with specific sequence of RNAi. Future work should focus on siRNAs from conserved regions of other PERV genes.     

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.     

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.     

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.     

Xenotransplantation and porcine cytomegalovirus

Porcine microorganisms may be transmitted to the human recipient when xenotransplantation with pig cells, tissues, and organs will be performed. Most of such microorganisms can be eliminated from the donor pig by specified or designated pathogen‐free production of the animals. As human cytomegalovirus causes severe transplant rejection in allotransplantation, considerable concern is warranted on the potential pathogenicity of porcine cytomegalovirus (PCMV) in the setting of xenotransplantation. On the other hand, despite having a similar name, PCMV is different from HCMV. The impact of PCMV infection on pigs is known; however, the influence of PCMV on the human transplant recipient is unclear. However, first transplantations of pig organs infected with PCMV into non‐human primates were associated with a significant reduction of the survival time of the transplants. Sensitive detection methods and strategies for elimination of PCMV from donor herds are required.     

Molecular characterization of the porcine endogenous retrovirus subclass A and B envelope gene from pigs

Xenotransplantation of porcine organs has the potential to overcome the current critical shortage of allogenic organs for transplantation in humans. However, the existence of porcine endogenous retroviruses (PERVs) presents a problem for the clinical use of xenografts from pigs. In an attempt to understand the molecular characteristics of PERVs, we cloned the PERV env gene from six pig breeds (ie, Berkshire, Duroc, Landrace, Yorkshire, and two types of miniature pigs) in Korea. A total of 141 env clones were isolated and their sequences were analyzed. Phylogenetic analyses of these genes revealed the presence of PERVs, from both classes A and B, in 54% and 46% of the env clones, respectively. Among these clones, 37 isolates had the correct open reading frame (ORF; 27 clones in subclass A and 10 clones in subclass B), while the others had premature termination. These PERV nucleotide sequences can be used in a database for comparisons of PERV distribution among different pig breeds and for monitoring PERV infection using isolates with functional ORFs. Recombinant envelope of subclass A and B with functional ORF was expressed by vaccinia virus systems. Additionally isolated env clones can be used for various experiments, such as PERV control and infectivity tests, and may enhance the understanding of molecular mechanisms through pseudotyped PERV viruses.     

Phylogenetic analysis of porcine endogenous retroviruses expressed in Chinese pigs based on envelope sequences

The promise of successful clinical xenotransplantation is now offset by the potential risk of transmission of porcine endogenous retrovirus (PERV). PERV consists of three subtypes according to the varieties of env sequences. We analyzed PERV subtypes in two species of Chinese pigs (Banna minipig inbred, BMI, and Wu-Zhi-Shan pig, WZSP). Positive A and B were detected while positive C was absent in the analyzed Chinese pigs. The polymerase chain reaction products were then cloned into a pGEM-T vector system and sequenced. Phylogenetic trees were constructed from the translated amino acids of PERVs and other type C and type D retrovirus, as well as the lentivirus in the GeneBank. The results suggested that PERV-A and PERV-B that exist in Chinese pig genomes share similarities with other PERV from the GeneBank and some type C retroviruses, including lymphotropic, leukemic and endogenous retroviruses.     

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 2a: source pigs—preventing xenozoonoses

Chapter 2 of the original consensus statement published in 2009 by IXA represents an excellent basis for the production of safe donor pigs and pig‐derived materials for porcine islet xenotransplantation. It was intended that the consensus statement was to be reviewed at interval to remain relevant. Indeed, many of the original salient points remain relevant today, especially when porcine islet xenotransplantation is performed in conjunction with immunosuppressants. However, progress in the field including demonstrated safe clinical porcine xenograft studies, increased understanding of risks including those posed by PERV, and advancement of diagnostic capabilities now allow for further consideration. Agents of known and unknown pathogenic significance continue to be identified and should be considered on a geographic, risk‐based, dynamic, and product‐specific basis, where appropriate using validated, advanced diagnostic techniques. PERV risk can be sufficiently reduced via multicomponent profiling including subtype expression levels in combination with infectivity assays. Barrier facilities built and operated against the AAALAC Ag Guide or suitable alternative criteria should be considered for source animal production as long as cGMPs and SOPs are followed. Bovine material‐free feed for source animals should be considered appropriate instead of mammalian free materials to sufficiently reduce TSE risks. Finally, the sponsor retention period for archival samples of donor materials was deemed sufficient until the death of the recipient if conclusively determined to be of unrelated and non‐infectious cause or for a reasonable period, that is, five to 10 yrs. In summary, the safe and economical production of suitable pigs and porcine islet xenograft materials, under appropriate guidance and regulatory control, is believed to be a viable means of addressing the unmet need for clinical islet replacement materials.     

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.