Porcine cytomegalovirus (PCMV), that is actually a porcine roseolovirus (PRV), is a common herpesvirus in domestic pigs and wild boars. In xenotransplantation, PCMV/PRV has been shown to ...significantly reduce the survival time of pig kidneys and hearts in preclinical trials with different non-human primates. Furthermore, PCMV/PRV has been transmitted in the first pig to human heart xenotransplantation and contributed to the death of the patient. Although transmitted to the recipient, there is no evidence that PCMV/PRV can infect primate cells including human cells. PCMV/PRV is closely related to the human herpesviruses 6 and 7, and only distantly related to the human CMV (HCMV). Antiviral drugs used for the treatment of HCMV are less effective against PCMV/PRV. However, there are well described strategies to eliminate the virus from pig facilities. In order to detect the virus and to eliminate it, highly sensitive detection methods and the knowledge of how, where and when to screen the donor pigs is required. Here, a comparative testing of organs from pigs of different ages using polymerase chain reaction (PCR)-based and immunological methods was performed. Testing young piglets, PCMV/PRV was detected effectively by PCR in blood, bronchoalveolar lavage fluid, tonsils and heart. In adult animals, detection by PCR was not successful in most cases, because the virus load was below the detection limit or the virus was in its latent stage. Therefore, detection of antibodies against selected recombinant proteins corresponding to epitopes detected by nearly all infected animals in a Western blot assay is advantageous. By contrast, immunological testing is not beneficial in young animals as piglets might have PCMV/PRV-specific antibodies obtained from their infected mother via the colostrum. Using a thoughtful combination of PCR-based and immunological methods, detection of PCMV/PRV in donor pigs for xenotransplantation is feasible and a controlled elimination of the virus by early weaning or other methods is possible.
Abstract 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.
Abstract Human endogenous retroviruses (HERVs) have been shown to be important in physiological and pathophysiological processes in humans. Several HERVs have been found to be expressed in the ...placenta—a tissue with special immunomodulatory functions that is responsible for nutrition of the embryo and the ability of the semiallogenic trophoblast to invade. The envelope proteins of HERV-W (also known as syncytin 1) and HERV-FRD (syncytin 2) were shown to be involved in cell fusion leading to the generation of the syncytiotrophoblast. Syncytin 2 was further shown to have immunosuppressive properties. Herein we analyse the expression of another HERV, HERV-K, which is characterised by open reading frames for all viral genes. Using immunohistochemistry and Western blot analysis, expression of the transmembrane envelope (TM) protein of HERV-K was studied in normal placental and decidual tissues obtained at different gestational ages. The TM protein was expressed exclusively in villous (VT) and extravillous cytotrophoblast (EVT) cells, but not in the syncytiotrophoblast or other cells. The expression of the TM protein of HERV-K in EVT cells was confirmed by Western blot analysis of isolated c-erbB2-expressing cytotrophoblast cells. Thus, this is the first report showing expression of the TM protein of HERV-K in normal human placental tissue with an exclusive expression in cytotrophoblast cells, suggesting a potential involvement of HERV-K in placentogenesis and pregnancy. Since retroviral TM proteins including the TM protein of HERV-K have immunosuppressive properties, expression of the TM protein of HERV-K may contribute to immune protection of the fetus.
There is a need to carefully consider the potential infectious risks associated with xenotransplantation. Whereas known viruses can easily be eliminated from donor pigs by designated pathogen free ...breeding of the animals, this is not possible for porcine endogenous retroviruses (PERVs) integrated into the genome of all pigs and unknown viruses. PERVs infect human cells in vitro and may theoretically like many retroviruses induce immunodeficiencies and/or tumours.
Recently a xenotropic murine leukaemia virus‐related virus (XMRV), a gammaretrovirus that is closely related to murine leukaemia viruses as well as to PERV, has been detected in human patients with prostate carcinoma, chronic fatigue syndrome (CFS) and also in a small percentage of clinically healthy individuals. Whereas several studies showed a broad distribution of XMRV in the USA, similar studies in Europe failed to detect this virus in the human population (for review see 1). These results clearly indicate that XMRV is not causally associated with prostate carcinoma and CFS. Either the virus is common in the USA (may be there are specific populations of rodents releasing this virus) and not in Europe, or it is a laboratory contamination.
If XMRV is indeed circulating in the human population, it has important implications for xenotransplantation. A test should be developed to discriminate between PERV and XMRV and the potential for recombination between the two viruses should be investigated. Recombination between the human tropic PERV‐A and the ecotropic PERV‐C has been described and recombinant PERV‐A/C was characterized by increased replication titers. Whether XMRV and PERV recombine remains unclear, however co‐packaging and pseudotyping between PERV and murine retroviruses have been described. This raises new questions: Should the xenotransplant recipient be pre‐screened for XMRV to avoid recombination? What measures can be taken when XMRV infection is detected in such a screen?
However, before dealing with these specific details, it is necessary to address the important broad questions concerning the distribution of XMRV and its impact on human health.
Reference
1. Denner J. Detection of a gammaretrovirus, XMRV, in the human population: Open questions and implications for xenotransplantation. Retrovirology 2010; 7: 16.
The zoonotic transmissions from non‐human primates of the human immunodeficiency virus (HIV‐1) that initiated the AIDS pandemic and the spread of many emerging infectious diseases teaches us that ...there is a need for carefully consider the potential risks associated with xenotransplantation. Whereas known viruses can easily be eliminated from donor pigs by designated pathogen free (DPF) breeding of the animals, this is not possible for porcine endogenous retroviruses (PERVs) that are integrated into the pig genome. Two such viruses, PERV‐A and PERV‐B, are present in all pigs and are able to infect human cells, whereas PERV‐C is not ubiquitous and only infects pig cells. In addition to these viruses, recombinant PERV‐A/C viruses were recently described 1‐3 that are able to infect human cells, are characterised by very high titre replication 4 and whose proviruses have been found de novo integrated in the DNA of somatic pig cells, but not yet in the pig germ line. However, since the receptor for PERV‐A/C is expressed on germ cells, infection of these cells is a theoretical possibility. Infection and integration of PERV‐A/C into the DNA of oocytes or sperm cells may lead to an endogenisation of the recombinant virus and transmission to offspring. PERV‐A/C viruses were also found in pigs with a very high incidence of melanomas although in this case the proviruses were found integrated in immune cells but not in tumour cells or in the germ line 5.
The risk posed by PERV‐A/C recombinant viruses for xenotransplantation could theoretically be eliminated by using pigs free of PERV‐C, as this would preclude recombination with PERV‐A 6. However, when the incidence of PERV‐C in different pig strains and multitransgenic pigs was evaluated, 97.2% were found to have PERV‐C in their germ line 7. To help overcome difficulties in obtaining animals free of PERV‐C, transgenic pigs were generated that express siRNA able to reduce expression of PERV‐A, PERV‐B and PERV‐C by RNA interference 8. This should contribute to a low expression all PERV proviruses and therefore to a lower risk of recombination.
Supported by Deutsche Forschungsgemein‐schaft, DFG, DE729/4.
References
1. Wilson CA, Wong S, Van Brocklin M, Federspiel MJ. Extended analysis of the in vitro tropism of porcine endogenous retrovirus. J Virol 2000; 74: 49–56
2. Wood JC, Quinn G, Suling KM, Oldmixon BA, Van Tine BA, Cina R, Arn S, Huanq CA, Scobie L, Onions DE, Sachs DH, Schuurmann HJ, Fishman JA, Patience C. Identification of exogenous forms of human‐tropic porcine endogenous retrovirus in miniature swine. J Virol 2004; 78: 2494–2501.
3. Bartosch B, Stefanidis D, Myers R, Weiss R, Patience C, Takeuchi Y. Evidence and consequence of porcine endogenous retrovirus recombination. J Virol 2004; 78: 13880–13890.
4. Denner J, Specke V, Thiesen U, et al. Genetic alterations of the long terminal repeat of an ecotropic porcine endogenous retrovirus (PERV) during passage in human cells. Virology 2003; 314: 125–133.
5. Dieckhoff B, Puhlmann J, Büscher K, Hafner‐Marx A, Herbach N, Bannert N, Büttner M, Wanke R, Kurth R, Denner J. Expression of porcine endogenous retroviruses PERVs in melanomas of Munich miniature swine MMS troll. Vet Microbiol 2007; 123: 53–68.
6. Denner J. Recombinant porcine endogenous retroviruses (PERV‐A/C): a new risk for xenotransplantation? Arch Virol 2008; 153: 1421–1426.
7. Dieckhoff B, Kessler B, Jobst D, Kues W, Petersen B, Pfeifer A, Kurth R, Niemann H, Wolf E, Denner J. Distribution and expression of porcine endogenous retroviruses (PERV) in multi‐transgenic pigs generated for xenotransplantation. Xenotransplantation 2009; 16: 64–73.
8. Dieckhoff B, Karlas A, Petersen B, Kues WA, Kurth R, Niemann, Denner J. Knockdown of porcine endogenous retrovirus (PERV) expression by PERV‐specific shRNA in transgenic pigs. Xenotransplantation 2008; 15: 36–45.
Abstract
Solid organ and cell transplantation, including pancreatic islets constitute the treatment of choice for chronic terminal diseases. However, the clinical use of allogeneic transplantation is ...limited by the growing shortage of human organs. This has prompted us to initiate a unique multi-center and multi-team effort to promote translational research in xenotransplantation to bring xenotransplantation to the clinical setting. Supported by the German Research Foundation, an interdisciplinary group of surgeons, internal medicine doctors, diabetologists, material sciences experts, immunologists, cell biologists, virologists, veterinarians, and geneticists have established a collaborative research center (CRC) focusing on the biology of xenogeneic cell, tissue, and organ transplantation. A major strength of this consortium is the inclusion of members of the regulatory bodies, including the Paul-Ehrlich Institute (PEI), infection specialists from the Robert Koch Institute and PEI, veterinarians from the German Primate Center, and representatives of influential ethical and religious institutions. A major goal of this consortium is to promote islet xenotransplantation, based on the extensive expertise and experience of the existing clinical islet transplantation program. Besides comprehensive approaches to understand and prevent inflammation-mediated islet xenotransplant dysfunction immediate blood-mediated inflammatory reaction (IBMIR), we also take advantage of the availability of and experience with islet macroencapsulation, with the goal to improve graft survival and function. This consortium harbors a unique group of scientists with complementary expertise under a cohesive program aiming at developing new therapeutic approaches for islet replacement and solid organ xenotransplantation.
Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig‐to‐primate xenotransplantation. Here, we report the generation of pigs with decreased ...tissue factor (TF) levels induced by small interfering (si)RNA‐mediated gene silencing. Porcine fibroblasts were transfected with TF‐targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild‐type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF‐α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi‐transgenic pigs for xenotransplantation.
Pigs with siRNA mediated knockdown of tissue factor show improved characteristics of blood coagulation and are thus useful for xenotransplantation.
A total of 335 infectious diseases was reported in the global human population between 1940 and 2004, the majority of which were caused by zoonotic pathogens 1. Although viral pathogens constitute ...only 25%, some have spread worldwide with most starting from Central Africa. These include human immunodeficiency virus (HIV) causing acquired immunodeficiency syndromes (AIDS), chikungunya virus and West Nile virus, which also cause severe diseases in humans. HIV‐1 and HIV‐2, for example, are the result of trans‐species transmission from non‐human primates 2 to humans sometime in the last century. The spread of two henipaviruses causing fatal diseases in horses, pigs and humans has been observed in Asia and Australia, and although these viruses represent transspecies transmissions from bats, secondary transmissions from pigs to humans have also occurred. These and many other examples of emerging infectious diseases call for strong safety considerations in the field of xenotransplantation. Whereas known viruses can easily be eliminated from donor pigs, strategies should be developed to detect new zoonotic pathogens. In addition, all pigs carry porcine endogenous retroviruses (PERVs) in their genome. Two of these, PERV‐A and PERV‐B, as wells as recombinant PERV‐A/C are able to infect human cells. The greatest threat appears to come from the recombinant PERV‐A/C viruses as they appear to have an increased infectivity 3,4. An increase in PERV expression was not observed in multitransgenic pigs expressing DAF, TRAIL and HLAE, generated to prevent immune rejection 5. Our laboratory has developed a variety of strategies to prevent PERV transmission following xenotransplantation: (i) selection of animals that do not harbour PERV‐C genomes in order to prevent recombination, (ii) selection of PERV‐A and PERV‐B low‐producers 6, (iii) development of an antiviral vaccine to protect xenotransplant recipients 7 and (iv) generation of transgenic pigs in which PERV expression is inhibited via RNA interference. Inhibition of PERV expression using either synthetic small interfering (si) RNA or short hairpin (sh) RNA was demonstrated in PERV infected human cells 8, in primary pig cells 9 and in all transgenic piglets born 10. A second generation of pigs expressing PERV‐specific siRNA is now under study and experiments have been started to introduce multiple shRNA.
Supported by Deutsche Forschungsgemeinschaft, DFG, DE729/4.
Porcine endogenous retroviruses (PERVs) infect human cells in vitro and therefore represent a risk for xenotransplantation. However, first clinical transplantations of pig cells into humans or ex ...vivo perfusions did not result in transmission of PERVs. On the other hand, recent experiments with SCID mice demonstrated infections with PERV in vivo. In order to define and characterize human target cells, we studied numerous primary human cells and cell lines. Infection with PERVs was shown for human peripheral blood mononuclear cells, primary endothelial cells, and primary aortic smooth muscle cells as well as lymphocytic, monocytic, and epithelial cell lines.
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