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Xenotransplantation: is the risk of viral infection as great as we thought?
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MOLECULAR MEDICINE TODAY, MAY 2000 (VOL. 6)
Xenotransplantation: is the risk of viral infection as great as we thought? Walter H. Günzburg and Brian Salmons
Two major hurdles remain before xenotransplantation can enter the clinic. The first is the more technical issue of being able to overcome the human immune response that leads to rejection of transplanted organs/cells from other species. The second, reviewed here, concerns the potential risk of inadvertent transfer of animal viruses present in the xenotransplant that are able to infect the human recipient. The threat from viruses is a particularly contentious topic because it poses a risk not only to those individuals who receive xenotransplants, but also to healthy individuals who come into contact, either directly or indirectly, with the xenotransplant recipient. In this review, we describe some of the virus types, in addition to the much discussed porcine endogenous retroviruses that might cross the species barrier, and assess the risk of such viruses causing disease in human hosts. THE rationale for investigating the feasibility of xenotransplantation includes: (1) the shortage of organ donors; (2) the avoidance of disease; and (3) the possibility of genetically engineering transplanted material. However, a number of hurdles, including: (1) the immune response to the xenograft; (2) differences in physiology between the donor xenograft and the recipient; (3) the production of suitable organs for xenotransplantation; and (4) the risk of infection to the patient and to society stand in the way of the routine application of xenotransplantation.
The infection risk in conventional allotransplantation Conventional allotransplantation has always been associated with the risk of transferring potentially infected material from the donor to the Walter H. Günzburg* PhD Professor and Chairman Institute of Virology, University of Veterinary Sciences, Veterinärplatz 1, A-1210 Vienna, Austria. Tel: 143 1 25077 2301 Fax: 143 1 25077 2390 *e-mail: [email protected] Brian Salmons PhD Scientific Director Bavarian Nordic Research Institute, Fraunhoferstr. 18b, D-82152 Martinsried, Germany.
transplant recipient. Although it is currently possible to screen for a wide variety of known pathogens, the risk remains, and both known and unknown microorganisms have been, and are still, unwittingly transferred. Obvious examples of known agents that are transferred to allotransplant recipients include the hepatitis viruses and HIV. A number of circumstances favour the risk of infection during allotransplantation. These include the urgency with which organs and tissues must be transplanted, often from donors who have only very recently, and without sufficient time for a thorough investigation, entered that status. Vascularization of the graft and direct cell–cell contact also contributes to the risk of transmission of infectious agents. Additionally, the heavy immunosuppression required for allotransplantation favours the establishment of many infections that would normally be easily dealt with by the immune system of the patient.
The infection risk in xenotransplantation Many of the problems associated with allotransplantation should be solved by xenotransplantation because standardized, well-characterized donor organs or tissues will be available ‘off the shelf’. However, a new problem of xenozoonosis might arise that, in contrast to the infections outlined above (which directly affect only the transplanted individual and close partners) could affect the patient’s family, friends, healthcare staff and, ultimately, the whole of society. It is because of this potential danger that the risk of xenozoonosis represents one of the most debated aspects of xenotransplantation. Only viruses will be considered in this article, because they represent a huge reservoir of potentially untreatable infections. Most known viruses can be excluded using a well-controlled specific pathogen free (SPF) environment. However, a few of the known viruses – specifically, those that integrate into the genome of the infected cell and do not
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lead to lysis of the cell – as well as the incalculable number of unknown viruses, pose a major threat.
Xenotransplantation compromises anti-virus defence systems The process of genetically engineering xenotransplantation donors (mainly pigs) so that their organs will not immediately be rejected upon transplantation into humans, concomitantly compromises the first line of defence of the human recipients against cross-species virus attack. Under normal circumstances, enveloped viruses, even if they are present in xenotransplanted material, would be rapidly eliminated by complement-mediated lysis (Box 1). One current goal, to prevent the rapid destruction of xenotransplants, is the inhibition of the complement system, for instance, by expressing complement regulatory proteins in the xenograft (Box 1). However, these regulators will also inactivate the highly effective virolysis system – which will no longer provide adequate protection against zoonotic viral infection (Fig. 1). An additional problem is raised by the circumvention of the physical barriers normally restricting entry. Usually, viruses must gain entry to a new host by overcoming physical barriers, such as the mucosal surfaces or the skin, without losing their infectivity. In the case of xenotransplantation, however, these barriers are effectively overcome by the direct implantation of potentially virus-harbouring material in the human, in such a way that it will be directly bathed by blood and other body fluids. The combination of SPF breeding conditions and rigorous testing guarantees that most of the known viruses are eliminated from organs before xenotransplantation. In these cases, the loss of the virolysis system and the lack of physical barriers would just mean the loss of a
Box 1. Control of hyperacute rejection of non-primate organs Non-primates, such as pigs, possess the enzyme a-1,3-galactosyl transferase (a-gal), and many of their structural proteins carry a-gal modifications. By contrast, humans and old-world primates do not express agal, but have natural antibodies (XNAs) directed against normal gut flora that cross-react with such a-gal modifications present on non-primate proteins. Thus, normal non-primate organs used for transplantation display a-gal residues on their surface, which, upon transfer into human recipients, are recognized by XNAs – triggering the activation of the complement system and leading to the hyperacute rejection reaction. A number of strategies have been used to eliminate the hyperacute reaction against transplanted non-primate organs. Circulating XNAs can be removed from the blood of a xenotransplant recipient. Alternatively, complement can be inhibited using monoclonal antibodies. Others have attempted elective depletion of complement by substances such as cobra venom factor, which can substitute for one of the members of the complement pathway but is not subject to feedback regulation, resulting in complement depletion. A number of regulators of complement are also known: they include; decay accelerating factor (DAF); membrane cofactor (MCP or CD46); factor H; and CD59. One of the most popular strategies involves using transgenesis to engineer the expression of one or more of these regulators on the surface of organs to be transplanted and thus protect them from complement-mediated lysis.
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backup safety system. However, there are circumstances in which the lack of these two natural safety systems might have grave consequences. The most serious of these are the persistent viruses (which either integrate or episomally maintain their genetic information without necessarily lysing the host cell), unknown viruses or those that cannot be detected using current test methods (and thus present a potential, unpredictable risk in xenotransplantation).
Persistent virus infection Persistent infections can be defined as those in which virus remains in specific cells rather than being cleared by the immune response. Indeed, immunosurveillance is often responsible for the maintenance of the persistent state. Well-known examples include cytomegalovirus (CMV) or Epstein–Barr virus (EBV); infection lies undetected until immunosurveillance breaks down as a result of immunosuppression – for example, during transplantation or during the later stages of AIDS – whereupon these viruses become clinically relevant. Other persistent infections are characterized by a progressive disease; well-known examples include HIV, its animal forms (SIV, FIV etc.) and other animal retroviruses, such as equine infectious anaemia1 and visna2 virus. However, non-retroviruses such as the measles virus can also potentially cause persistent infections, such as subacute sclerosing panencephalitis3. Chronic infections are those in which a balance has been attained. Either the rate of viral replication and cell destruction equals the production of new uninfected cells, or all cells are infected but there is no cell killing, so both viral- and cell-replication occur at the same time. Many persistent viruses escape elimination by the host immunemediated defence mechanisms by modulating or even evading the host immune system. Measles virus, for instance, restricts the expression of viral antigens in subacute sclerosing panencephalitis; HIV shows extreme antigenic variation in AIDS; CMV and adenoviruses both downregulate the expression of MHC class II; and CMV also alters the production of lymphocyte adhesion (ICAM-1, LFA-1) or co-stimulatory (B7) molecules. Another strategy followed by viruses such as HIV, herpesviruses (such as EBV) and poxviruses is to modulate the production of cytokines by the cell, and many viruses (for example, influenza) have specific strategies to downregulate the production of the classical antiviral, interferon. In all these scenarios, a change of host can upset the delicate balance between virus and cell replication, or change the pathogenicity profile of the virus. Prolonged incubation periods might, however, remain a feature of these infections, making early detection difficult. It is not clear whether the immune modulatory strategies practised by many viruses will still be active after trans-species transmission of a virus, or whether, with time, there will be a breakthrough of a frank viral disease.
Persistent retroviral infections Probably the most important, and certainly the best-understood, category of persistent viruses are the retroviruses. These enveloped viruses have an RNA genome that is reverse transcribed into a doublestranded DNA ‘proviral’ form after infection of the target cell. The proviral DNA is integrated in a random fashion into the host-cell DNA and persists for the life span of the cell. For instance, HIV persists in infected individuals despite aggressive highly active anti-retroviral chemotherapy (HAART) regimes (also known as triple therapy), because of the proviral state. If a retrovirus infects a host germ-cell, the proviral information becomes part of the genetic information of the offspring, and is termed an endogenous retrovirus. Thus, persistence of
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Figure 1. Xenotransplantation eliminates the antiviral defence system. (a) Enveloped viruses, such as retroviruses, arising from non-primate cells incorporate part of the plasma membrane of the cell from which they bud. Consequently, they display proteins bearing the a-1,3 galactosyl transferase (a-gal) modification on the surface of virions, and are recognized by xenoreactive natural antibodies (XNAs) present in human serum, triggering complement-mediated lysis or virolysis. This figure shows the viral RNA being packaged by viral core proteins and budding from the producer cell. Budding involves the core moving through the plasma membrane and becoming coated with this membrane. Viral surface-proteins present in the membrane, as well as a-gal modifications, will therefore be displayed on the surface of the viral particle. XNAs generated against crossreacting human gut-flora react with the a-gal modifications and trigger complement mediated lysis (virolysis) of the virus particle49. (b) Enveloped viruses, such as retroviruses, arising from transgenic xeno-organs engineered to express complement regulators (to prevent hyperacute rejection) will also incorporate these complement regulators when they bud through the plasma membrane. Expression of these complement regulatory proteins on the surface of the budding virion allows it to escape virolysis28,50.
viral genetic information is not restricted to the infected animal but is found in every successive generation. Like any other cellular gene, the integrated provirus is transcribed by the cellular machinery, and the resultant RNA used both for translation into viral structural proteins and as a genome for the resultant retroviral progeny4. Retroviruses are associated with tumour induction, immunodeficiencies and neurodegenerative diseases although often only by an indirect mechanism4. Retroviruses have, in the past, been known to jump species. It is now generally accepted that SIV jumped from infected monkeys to humans giving rise to HIV in the 1930s5–7. Because of the endogenous nature of other retroviruses, it is also possible to analyse the sequence relatedness of these ancient proviruses. Recently, the sequences of endogenous retroviruses related to murine leukaemia virus (MLV) have been compared in a number of different species. Phylogenetic analysis revealed that viruses isolated from a particular host generally cluster together, suggesting that horizontal transmission of infectious virus between species rarely occurs. Nevertheless, evidence was found for two instances of cross-species transmission; one from mammals to birds, which was followed by rapid spread to other avian hosts; the other involved the transmission of a gibbon ape leukaemia (GaLV) retrovirus to koala bears, possibly via rodents8, although previously it has been speculated that this virus was originally transmitted from mice to gib-
bons9,10. Disturbingly, both GaLV and the koala virus are closely related to porcine endogenous retroviruses (PERVs; see below)8.
Persistent herpesvirus infections The persistence of a virus can also result from the establishment of a latent infection, as in the case of herpesvirus infection, in which virus cannot be detected between episodes of recurrent disease. These viruses are extremely problematic, even in conventional allotransplantation. Herpesviruses are enveloped viruses with a DNA genome that can be maintained latently (in other words, non-expressed) for the life of the host cell as a circular episomal form. The latent form of the virus is often transmitted from the donor to the organ/tissue recipient. Not only might such latent herpesvirus infections persist undetected for long periods before being activated by stress or environmental factors, but reactivated herpesviruses, which cause death of the host cell during their lytic cycle, also frequently show an increased pathogenic profile after cross-species transfer, leading to highly virulent forms of disease11. As pigs are the most likely potential xenograft donors, the search for potentially zoonotic herpesviruses is on. Until recently, only two herpesviruses that infect pigs were known: pseudorabies virus, an aherpesvirus, which causes encephalomyelitis and respiratory tract inflammation12; and porcine cytomegalovirus, a b-herpesvirus, found in 201
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the respiratory tract of pigs, which causes atrophic rhinitis13. Recently, however, two g-herpesviruses have been discovered (porcine lymphotropic herpesvirus types 1 and 2)14 and it seems that other herpesviruses that have yet to be detected might be present in some pigs.
Other persistent viral infections Besides these viruses, there are other viruses that show abnormally long time lags between the infection event and viral replication and spread through the body. One such well known virus is the rabies rhabdovirus11, and other as yet unknown members of the rhabdovirus family might show similar biological properties. Other animal viruses, the human counterparts of which have the potential to cause debilitating chronic infections, such as hepatitis (members of a number of virus families) or neoplasia (papilloma or polyoma viruses) are also of concern. Although it would be possible to screen for known persistent viruses in organ donors (Box 2), as yet unknown viruses probably also exist. The most dangerous of these are viruses that have co-evolved with their host – so there is an undetected or chronic infection that does not produce disease in the natural host. However, potentially long latency periods after transfer to the human recipient, coupled with possibly altered pathogenicity profiles, mean that these viruses, and the resulting illnesses, can only be determined after the event. Time and time again, new examples arise of viruses that apparently have existed in perfect symbiosis with their natural animal host for many years, and thus have not been recognized owing to their lack of pathogenic phenotype, but that cause human disease upon cross-species transmission. Examples of such viruses classically include Ebola and Marburg virus disease, SIV/HIV, and most recently Hanta, Hendra and Nipah viruses. Interestingly, these viruses are all enveloped viruses transmitted from animals such as non-human primates (Ebola, Marburg, SIV/HIV), rodents (the Sin Nombre Hanta virus15), bats (Hendra16,17) and pigs (Nipah18), although fruit-eating bats are probably a reservoir of Nipah virus. In these cases, even with an intact virus defence system, these zoonotic enveloped pathogens were quickly able to establish themselves in humans and manifest their pathogenicity. At least for some of these viruses, subsequent human-to-human spread has
Box 2. Molecular methods for screening for new zoonotic agents A number of molecular techniques are now available for the very sensitive detection of viral genetic information. They are usually based on a polymerase chain reaction (PCR) and include conventional DNA- and reverse transcriptase-PCR, nested PCR and more sophisticated PCRbased methods such as Taqman Real-Time PCR and in situ PCR. However, these techniques are sensitive to small variations in genome sequence – raising the possibility of false negatives47. An additional problem, not often discussed, but highly relevant to the prevention of false negatives, is the correct and reproducible pre-assay sample preparation and nucleic acid extraction; multiplex PCR techniques have been developed to address this48. Thorough validation regimes are required to ensure the quality of the PCR results obtained. Unknown viruses can only be traced by their similarity at the nucleic acid level to other viruses in their family, using redundant PCR approaches. This puts severe constraints on the detection systems because the degree of nucleic acid homology between individual viruses of the same family can vary considerably.
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been documented (Ebola, HIV). However, it appears that this has only been observed after initial zoonotic transfer from non-human primates. There is no evidence for human-to-human spread of zoonotic viruses of non-primate origin, although this has been extensively studied for the rodent-originating Sin Nombre virus19,20. All these viruses were unknown before the outbreaks of disease they caused in humans. It would be unwise to assume that there are no more of these human pathogenic viruses lurking in the animal world.
Pigs as potential organ donors Both physiological and ethical reasons suggest that the majority of, if not all, xenotransplants will be obtained from pigs. Non-human primates could represent a source of organs for babies and very young children but, in these circumstances, it might even turn out that the immunological problems facing these special situations can be overcome without resorting to transgenesis. A whole range of potentially zoonotic porcine viruses are known (Table 1). Also, viruses that are normally not regarded as zoonotic but that can replicate in human cells under laboratory conditions, as well as those that could recombine to form a potentially new type of virus, must be considered.
Porcine endogenous retroviruses Of all the potential porcine infectious agents, the porcine endogenous retroviruses (PERVs) have aroused the greatest interest. Assays for the presence of PERV DNA will always yield positive results because all examined pigs contain a number of these viruses integrated into their genomes. So far, PERV-A, PERV-B and PERV-C have been characterized. Analysis of pigs worldwide indicates that they contain 10–25 copies of PERV-A, around ten copies of PERV-B and either none or a variable number of copies of PERV-C at distinct integration loci21. There is no information currently available as to which of these loci produce infectious virus or potentially pathogenic viral proteins. PERV-C appears to be severely compromised in its ability to infect human cells, whereas relatively good infection titres have been obtained with PERV-A and -B (Ref. 22). Further passage of the virus produced from such infected human cells leads to increased infectivity of human cells in vitro23.
Spread of PERVs to humans What is the chance of these or other porcine viruses spreading to human recipients after xenotransplantation? In recent studies24, 160 patients who had been exposed to living porcine cells in the course of various treatment regimes for periods of up to 460 days were analysed. All patients tested negative for porcine virus transmission except for some of the patients who received extracorporeal splenic perfusion in Russia. These patients were in contact with porcine cells for 50–60 min. Of the 30 positive samples from the Russian group, 23 showed clear evidence of circulating pig DNA as determined by the presence of other porcine nucleic acids of mitochondrial or centromeric origin. This suggests the presence of porcine cells in these patients (chimerism) and, thus, it is possible that the positive PERV DNA signal was derived from integrated copies in these porcine cells, rather than from infection events. Not enough DNA was isolated to permit the analysis of the remaining seven samples. The chimerism suggested by the presence of mitochondrial or centromeric porcine DNA (microchimerism) was observed for up to 102 months. Others have described microchimerism specifically after spleen-cell transplantation into non-immunosuppressed and non-irradiated mice, which leads to lasting tolerance25, but for shorter periods. Paradis and
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Table 1. Some zoonotic and potentially zoonotic porcine viruses Transmission/ infection of human cells
Virus family
Virus name
Porcine disease
Remarks
Zoonotic
Orthomyxoviridae
Influenza
Influenza
Zoonotic
Paramyxoviridae
Nipah
Zoonotic
Flaviviridae
Japanese encephalitis (JE)
Zoonotic
Rhabdoviridae
VSV
Vesicular stomatitis
Zoonotic
Picornaviridae
Aptho
Foot and mouth disease
Zoonotic
Picornaviridae
Entero
Swine vesicular disease
Zoonotic Zoonotic Probably zoonotic
Rhabdovirus Poxviridae Parvoviridae
Lyssa Vaccinia Parvo (PPV)
Rabies Swinepox-like SMEDI-syndrome
Probably zoonotic
Herpesviridae
Aujeszky’s disease
Possibly zoonotic Possibly zoonotic
Poxviridae Picornaviridae
Pseudorabies (PRV) Suipox Cardio
Killed 20–40 million people (1918–1919; Spanish influenza). Possesses a segmented genome – virus contains eight pieces of RNA, each encoding a different gene. Reassortment results in new variants with new properties, e.g. reassortment between pathogenic porcine viruses and highly transmissible human viruses formed the H1N1 strain, which is lethal to humans Newly described in Malaysia: has killed over 60 adults. First described in workers exposed to pigs Fatal human disease transmitted by mosquitoes, with virus amplification in pigs and birds. JE virus exists in Asia, where swine raising is common. Infections in pigs are generally inapparent, except for stillbirth or abortion in pregnant swine, and aspermia in boars Found in many mammals, probably transmitted by mosquitoes and sandflies. Infected humans show transient symptoms of influenza Very few cases, disease is relatively benign in humans; subclinical or similar to animal symptoms Porcine variant of the human pathogen Coxsackievirus B, it causes a high degree of morbidity in infected animals and an influenza-like illness in humans Infects all warm-blooded animals and is almost invariably fatal Many original outbreaks of swinepox were caused by vaccinia A known contaminant of cell-culture trypsin and of blood products (Factor VIII). Can infect human cells, 50% of porcine islet recipients develop antibodies to PPV Wide host range, infects human cells in vitro. Recently, three patients seroconverted after previous contact with a cat that died of PRV Usually a mild disease with lesions restricted to the skin Wild rodents act as a reservoir for the virus. Tropism for the myocardium leads to rapid death in about 80% of infected cases Type 1 widely distributed in swine, but does not appear to be associated with any major disease symptoms. Type 2 has been identified as associated with post-weaning wasting disease syndrome in pigs. Antibodies to Type 1 have been identified in humans Widely distributed as endogenous viruses in the germline of pigs; originally isolated from a lymphosarcoma. Although expressed in a variety of porcine tissues, and capable of infecting human cells in vitro, there is no evidence for their replication and spread in humans Causes hepatitis and is closely related to human hepatitis E virus. Transmission to humans might be possible because the homologous human hepatitis E virus, which is associated with a 20% mortality rate in infected pregnant women, has been used to infect pigs Porcine polioencephalomyelitis Highly contagious viral disease of swine. The infection can run an acute (killing up to 90% pigs in a herd), subacute, chronic or inapparent course, mainly depending on the virulence of the virus. Antigenically related to bovine viral diarrhoea virus (BVDV) Transmissable gastronenteritis; vomiting and wasting disease; epidemic diarrhoea; respiratory disease Can infect swine PRRS causes abortion and premature birth as well as general fertility and respiratory problems
Swinepox Encephalomyocarditis
Potentially zoonotic Circoviridae
Circo
Post-weaning wasting syndrome
Potentially zoonotic Retroviridae
PERV
Lymphosarcoma?
Potentially zoonotic Floating genus
Hepatitis E
No evidence No evidence
Picornaviridae Flaviviridae
Entero Pesti
Teschen/Talfan disease Hog cholera
No evidence
Coronaviridae
Corona
Various
No evidence No evidence
Paramyxovirus Arteriviridae
Rinderpest Arteri
No evidence No evidence
Reoviridae Herpesviridae
Rota b herpes
No evidence
Asfarviridae
No evidence No evidence
Caliciviridae Flavivirus
African swine fever Vesi Pesti
Rinderpest Porcine respiratory and reproductive syndrome (PRRS) Diarrhoea Cytomegalovirus disease Widely distributed in swine and associated with inclusion body rhinitis and reproductive failure. No evidence that humans are infected, however, human CMV is a major problem in transplantation medicine African swine fever Limited to members of the Suidae – all attempts to infect members of other species have been unsuccessful Vesicular exanthema Eradicated Bovine viral diarrhoea Widely spread in cattle. The virus can also infect pigs, sheep and other ruminants leading to persistent lifelong infection and shedding
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Glossary Antigenic variation – Altered protein composition, which is recognized by the immune system. Antisense – Nucleic acid molecules generated by reversing the orientation of the transcribed region of a gene, which then forms a duplex with the natural ‘sense’ mRNA transcript, thereby preventing its translation. Autologous – Derived from the same individual. Chimerism – A state in which two or more genetically different populations of cells co-exist. Co-stimulatory molecules – Molecules that mediate interactions between immune cells. Cytokines – Intercellular signals, usually proteins, involved in the regulation of cellular proliferation and function. Interferon – Secreted molecule induced by virus replication that blocks protein synthesis and cell growth, and thus blocks viral multiplication. Lymphocyte adhesion molecules – A family of mammalian carbohydrate-binding adhesion molecules that mediate immune-cell interactions. MHC class II – A cluster of closely linked genetic loci encoding class II polypeptide products involved in the generation and regulation of immune responses. Provirus – A viral genome that is integrated into the genome of its host cell and transmitted to its progeny cells and, when integrated into the genome of a germ cell, from generation to generation. Pseudotyping – Pseudotyped or phenotypically mixed virions can arise when the same cell is simultaneously infected with two enveloped viruses. Such virions usually consist of the core and genome from one virus and the envelope from another. Reassortment – The process by which a novel virus arises from a cell that is co-infected with two viruses. It involves exchange of viral genetic information. Ribozymes – Catalytic RNA molecules that can promote specific biochemical reactions without the need for ancillary proteins – in this case the specific cleavage of viral RNA. Xenotransplantation – The transfer of organs or tissues from a donor animal of one species to the recipient of another species (e.g. pig to human). Xenozoonosis – A zoonotic infection that can be transmitted by the xenotransplantation of animal tissues or organs. Zoonotic infection – A disease that is transmissible from nonhuman vertebrates to humans.
colleagues speculate that the signal comes from porcine dendritic- or stem-cells that are only expressing low levels of a-l,3-galactosyl transferase (a-gal), thus allowing them to escape antibody-mediated clearance. Although it is known that human allotransplantation of solid 204
organs transfers passenger cells that might give rise to a state of persistent microchimerism26,27, this is after initial immunosuppression. Whether the observed PCR signals really originated from microchimerism, and whether sequencing would have provided more information, remain unanswered questions. In addition, this study did not reveal any evidence for pathogenicity mediated by other porcine viruses, even though some patients were followed up for periods of up to 102 months (eight and a half years). Although such events cannot be ruled out, they are probably very rare. However, neither of these findings are unexpected – what might happen when humans are exposed over long periods to modified porcine viruses that incorporate complement regulators that could disarm the human virolysis system28 remains unknown. Also, the study does not address issues relating to organ xenotransplantation, in contrast to islet and skin transplants or extracorporeal perfusion, where it is to be expected that both the time of exposure and the connection to the circulatory system will increase the amount of infectious virus that contacts the host.
Recombination of PERVs with human endogenous retroviruses It has been estimated that 1–5% of the human genome comprises retroviral or retrovirus-like sequences29. Most of these endogenous retroviruses represent partial retroviral genomes, often solitary long terminal repeats (LTRs) (Fig. 2), which themselves are not biologically active, although they can be transcribed into RNA. Although HERVs have not been shown to give rise to infectious retrovirus29, they do represent a potential source of material that could take part in recombination reactions with porcine retroviruses to create new, potentially pathogenic variants. Recombination in retroviruses is believed to occur mainly at the RNA level30 (Fig. 2) and is favoured by sequence homology. Recombination between non-homologous retroviral genomes is 100–1000-fold less likely than that between homologous ones31. Human and porcine retroviral sequences share little sequence homology8, thus the chance of recombinants arising is probably equal to that of recombination with any heterologous piece of RNA. By contrast, the chance of intra-species recombination of retroviral sequences to generate new viruses seems much higher.
The PERV-free pig: dream or reality? To eliminate the potential risks from PERVs that can infect human cells it will be important to remove those PERV loci that are biologically active from the porcine donors. If all the identified loci are biologically active, this would not be feasible. However, it seems more likely that only a few are biologically active, considering what is known about human32 and murine33 endogenous retroviruses; and if the active viruses could be identified it would be possible to inactivate them using current knockout technology. It will be difficult to conclusively evaluate which are biologically active, and it cannot be ruled out that the xenograft environment (for instance immunosuppression, or other drug treatments) might not activate a previously silent endogenous copy. It is therefore unrealistic to imagine that a PERV-free porcine organ donor can be produced in the foreseeable future. Nevertheless, a number of extra safety mechanisms, which are analogous to the safety devices used in gene therapy, could be built into the pig to ensure that, should one of the other PERVs become active for any reason, its expression could be controlled and eliminated. Such safety devices include the use of antisense technology to control PERV expression, ribozymes directed against PERV RNA, antivirals developed against points in the PERV life cycle and immunization to prevent infection.
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Figure 2. Retroviral recombination. Retroviruses are the only viruses with a diploid genome. This means that two copies of the viral RNA genome are packaged into the virion. The RNA genomes are identified and packaged by virtue of specific sequences known as packaging signals51. However, even in the absence of these signals, heterologous RNAs can be packaged52. If two disparate molecules of RNA are packaged into a virion, recombination can occur during reverse transcription. It is believed that the reverse transcriptase enzyme responsible for generating the DNA copy, which will become integrated into the target cell as the provirus, can jump from one RNA molecule to the other, copying a part of one and then juxtaposing the copy of the other molecule. This template-switching is most probably favoured by template homologies, although it appears that it can also take place in its absence. (a) A retrovirus infects a host cell by virtue of an interaction with a specific receptor. (b) The retrovirus is internalized, uncoated and the viral RNA genome is reverse transcribed to a DNA form. (c) The DNA form moves to the nucleus and integrates in the genomic DNA of the target cell. (d) The integrated provirus is then transcribed to generate viral RNA, which is used to make viral structural proteins, and is packaged into newly formed virions as their genetic information. Other endogenous retroviral sequences also present in the cell might be transcribed into RNA (e). If these RNAs carry the appropriate packaging signals (f), they too can be packaged into newly formed virions. Even retroviral RNAs lacking the correct packaging signals and non-retroviral RNAs can be packaged at a low frequency. (g) The retrovirus particle buds from the cell. Retroviruses carry two copies of their genetic information, so they can package two different RNAs, as shown here (white and grey). (h) The virus particle infects a second host cell and is internalized. (i) Recombination can occur during reverse trancription (see inset): (j) Viral DNA synthesis is primed by a tRNA molecule bound to the (white) viral RNA. DNA synthesis proceeds to the end of the template RNA. (k) As a result of the terminal duplications of the R sequence in the viral RNA, the nascent DNA strand may become translocated to the R region on the second (grey) RNA template. During further DNA synthesis (l) a second-strand jump from the grey RNA to the white RNA (m) will result in the generation of a hybrid DNA molecule carrying part of the white and part of the grey coding sequences. After integration and transcription of this provirus, new virus particles will emerge carrying the genetic information of the hybrid virus.
Other types of porcine viral recombinants It has long been known that when a cell is infected with two different viruses, novel viruses can emerge with new properties. The new viruses can result from: (1) recombination at the genetic level; (2) mixing of viral proteins (pseudotyping); or (3) mixing of viral genetic information (reassortment). These novel viruses are unpredictable and can
gain a novel pathogenic phenotype in the host; for instance, reassortment in influenza can lead to antigenically-shifted variants that cause severe public health problems. If the event occurs at the genetic level (recombination or reassortment), the change will be fixed over the next generations, whereas even pseudotyping can open the possibility of entry into new types of cells with subsequent secondary effects. 205
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Adaptation of porcine viruses to the human environment
Once a virus that originates in a non-primate (and thus has the a-gal modification) has replicated in a human cell, the progeny virions will no longer carry the a-gal modification and, thus, will no longer be sensitive to human complement-mediated virolysis. This adaptation of the virus to its new host usually results in an acceleration of the speed of successful replication. Viruses in general, and especially RNA viruses, show a high rate of mutation. Enhanced replication increases the chance of mutational change, leading to greater diversity in a virus population. This might favour the production and selection of a virus capable of efficiently infecting human cells. For instance, alterations in pathogenicity after cross-species transmission of SIV have been associated with mutation34. Infection is dependent on the presence of the appropriate receptor molecule on target cells. Nevertheless, viruses can be coerced into using new receptors. Current thinking would dictate the construction of transgenic porcine donors that express complement regulatory molecules in an attempt to regulate the hyperacute rejection reaction (Box 1). However, complement regulators such as CD46 or CD55 are natural receptors for measles35,36, echo37 and coxsackie B (Ref. 38) viruses. It is therefore possible that porcine viruses might evolve to use these receptors in the donor pig; after xenotransplantation, these viruses would be free to use these natural human molecules as receptors to enhance their spread through the patient.
Xenotransplantation and immunosuppression Finally, it should not be forgotten that, initially, xenotransplantation will probably require a more intense immunosuppression regime than currently practised for allotransplantation. Immunosuppression will increase the likelihood of a xenoinfection, giving viruses a better chance to adapt to their new environment. Furthermore, immunosuppression can result in the activation of persistent viruses, such as EBV and CMV, and might allow retrovirally-induced diseases to develop. Indeed, this is critical in models of retrovirus-mediated pathogenesis where new-born animals are inoculated in order to avoid the suppressive immune response.
Virological risks arising from xenotransplantation The virological risks associated with xenotransplantation can be divided into those affecting the patient and those affecting the community. In the light of probable medical benefit to the patient, the first risk can be negated in terms of the risk–benefit pay-off. The risk to the community, however, is of some concern. If the pathogenic phenotype of the porcine (or porcine–human recombinant) virus manifests itself early after transplantation, it might be possible to detect it early enough by a sophisticated screening procedure (Box 2). However, viruses causing no immediate disease will be very difficult to detect. Similarly, if at a later time (perhaps years later), the xenotransplanted patient becomes infected with a human virus that crosses into the xenotransplant and recombines with the porcine virus to generate a novel pathogenic virus, it will be practically impossible to identify until it is too late. Such a rare event could only be detected, and possibly prevented from spreading, by a life-long health status monitoring programme, and it will be extremely difficult to legislate for this remote possibility.
Risk assessment Can we put a figure on the likelihood that such events occur? This is, of course, very difficult. However, we can make some rough calculations that might help us to understand the potential size of the prob206
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lem. The number of known viruses that are zoonotic is small, thus the chance of an unknown virus proving to be zoonotic and readily transmitted in the human population is also small. Nevertheless, viruses, especially those with an RNA genome, can rapidly adjust to new environments, making the chance of adaptation, especially in the face of immunosuppressive regimes and the use of new receptors, quite likely. On the other hand, the virus that emerges will not necessarily be able to spread in the human population. Even if a virus adapts to the human cellular environment, can replicate there, and be transmitted from the xenotransplant recipient to the human population, it will rapidly elicit a controlling immune response.
Risk to the patient In one study, in which cells producing replication-competent MLV were infused into immunosuppressed non-human primates, lymphomas and viraemia were observed in three of 12 animals, and the animals died after around 200 days39. In these experiments, around 108 cells, titred in cell culture as producing at least 103 replication competent virus particles ml21, were introduced. Assuming that the in vitro titre was calculated from 106 producer-cells over a 24 h period, and assuming an in vivo virus half-life of around 4 min (as recently calculated40), the steadystate level of circulating infective virus particles should have been around 103 at any given time. However, titres of 104–105 infectious particles ml21 were measured in sera from animals developing lymphoma at the time of death. Since the plasma volume of the macaques used in these studies is calculated at about 30 ml kg21 (Ref. 40), and assuming the animals weighed around 10 kg, this would mean a total amount of infective virus of the order of about 107. Thus, in the three animals that developed lymphomas, the virus must have infected additional target cells, replicated and been released from these cells, thereby increasing the viral titre exponentially (<14 doublings of titre must have occurred over the 200-day period, compatible with an average doubling of titre every two weeks). Interestingly, all ten primates were subject to highdose total-body irradiation, and yet only three of the animals were unable to control the replication-competent virus infection. Seven of thes e animals were examined further. Of the five that controlled the infection, all showed serum antibody that was reactive against viral protein, whereas the two animals that developed lymphoma apparently failed to mount a humoral response to the virus. The finding that antibodies are produced after total-body irradiation seems surprising, but suggests that antibody production is possible even after severe immunosuppression. The data underline the importance of a humoral response in controlling infection in the autologous setting. Virus being produced from such autologously transplanted cells will not be subject to a-gal-triggered, complement-mediated virolysis. Thus, we might expect that virus arising from surface-modified transgenic xeno-organs should, in most cases, be controlled by a rapid antibody response, even in the immunosuppressed recipient. Given the more controllable nature of the xenotransplantation process, it might even be feasible to vaccinate recipients against known viruses, such as PERVs, before transplantation to ensure a good humoral anti-viral response. The data presented by Donahue and co-workers39 demonstrates, however, that replication-competent viruses can cause tumours in 30% of infected macaques under immunosuppressive conditions. It is not clear whether such viruses will be pathogenic in humans, indeed, other studies using both immunosuppressed and immunocompetent primates found no evidence of pathogenicity41,42. It is clear from human retroviral infections that there is no evidence for a common integration-site tumorigenic mechanism in patients infected with HIV-1, HIV-2, human T-cell
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The outstanding questions Are PERVs associated with disease in humans? • Do porcine cells harbour as yet unidentified zoonotic • viruses? porcine zoonotic viruses be transmitted from human• Will to-human? incorporation of immunomodulatory molecules en• Will hance viral pathogenicity? long will patients have to be monitored after xeno• How transplantation?
leukaemia virus-1 (HTLV-1) or HLTV-2. This is surprising, particularly for HIV-1, in light of the large number of patients infected, as well as recent calculations showing that 1012 virus particles per day are produced by such patients40. Thus, it could be argued that human retroviral infections contrast starkly with those of the animal world – where tumorigenesis by insertional mutagenesis is the rule, rather than the exception – a difference that might reflect the higher stability of the human genome. Indeed, the frequency of retrotransposon-induced disease-causing mutation in the human population has been estimated at 1:670, but none of these were the result of retroviral insertion43. From this point of view, it could be argued that PERV infection, even in the case of a productive infection, will not represent a great danger to the individual patient.
Risk to the germline Even the risk to the germline might be overstated in these patients. Although the use of condoms could protect against the spread of infectious agents, it has been argued that the prevention of procreation by xenotransplant recipients should also protect against the effects of harmful retrovirus-induced mutations in the human genome. However, it has been demonstrated that the human genome per se underlies an extremely high deleterious mutation rate – and yet the human population survives43,44. It is extremely unlikely that a rare integration into one germline cell will have a large influence on this.
or viruses that only cause symptoms after very long periods, could easily spread without being noticed. This can occur rapidly as has been documented for a feline parvovirus spreading to the dog population worldwide within a matter of years46.
Concluding remarks In conclusion, there is a certain level of risk of viral infection associated with xenotransplantation, and caution is warranted to prevent the emergence of new diseases, but it is difficult to quantitate these risks. In view of the enormous potential benefits associated with xenotransplantation, and the fact that no further progress can be made in the absence of further experimentation, it would be unwise to discontinue work in this area. The most probable candidates for viruses that could be a danger to society are those that show good infectivity and are difficult to contain, namely the persistent viruses with a long latency period. Thus, it is imperative that xenotransplanted patients are centrally registered and closely followed for health- and virus-status to detect emerging viruses over extended periods (Box 2). A battery of screening tests, based on our considerable knowledge of potentially zoonotic agents, as well as of physiological changes associated with viral infections in general, are essential to minimize potential risks to society, at least at present. Although this will not guarantee the detection and prevention of a new pathogenic virus, and although scenarios exist whereby the recombination might take place very late – even years – after transplantation, the health and virus screening programme should be conducted at least for the first five years after xenotransplantation. It will, however, be safe to assume that when xenotransplantation has become sufficiently established that patients survive for an average of five or more years, and when a sufficient number of patients (about 200) have been subjected to such a five-year screening programme without any signs of pathogenic virus release, the requirement for such post-operative screening could be readdressed. Acknowledgments. WHG would like to thank the Xenotransplantation Project Group of the Europäische Akademie Bad Neuenahr-Ahrweiler, Germany and their guests for stimulating discussions and support. The authors also gratefully acknowledge the constructive criticisms and suggestions of the reviewers.
Risks to society
References
What are the risks to the population at large? Uncontrolled virus production presents a theoretical risk to the entire human population – particularly to those in close contact with xenotransplant recipients. However, it should be remembered that retroviruses are, by nature, labile and are not transferred by aerosol (unlike other classes of virus that might play a role in xenotransplantation, such as influenza). They are susceptible to dehydration, can only be passed in body fluids and, even then, they are not very contagious45. Therefore, like HIV, their spread could be limited by health-control measures. More dangerous are novel aerosol-borne recombinants or human adapted viruses. Even in these situations, it should be possible to contain the virus quickly, provided symptoms develop rapidly. Recent scenarios in which rapidly fatal viruses, including Hendra, Nipah and Ebola, have been transmitted from animal hosts to humans reveal that, once recognized, spread of infection can be limited very quickly. Although it is conceivable that, with modern air transportation, such a viral disease might be transmitted to a large number of people before the symptoms are identified and the patient isolated, the speed of the outbreak of symptoms makes this unlikely, albeit not impossible. By contrast, non-pathogenic or weakly-pathogenic viruses,
01 Maury, W. (1998) Regulation of equine infectious anemia virus expression. J. Biomed. Sci. 5, 11–23 02 Pepin, M. et al. (1998) Maedi-visna virus infection in sheep: a review. Vet. Res. 29, 341–367 03 Liebert, U.G. (1997) Measles virus infections of the central nervous system. Intervirology 40, 176–184 04 Coffin, J.M. et al. (1997) Retroviruses, Cold Spring Harbor Laboratory Press 05 Gao, F. et al. (1999) Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 397, 436–441 06 Weiss, R.A. and Wrangham, R.W. (1999) From Pan to pandemic. Nature 397, 385–386 07 Corbet, S. et al. (2000) env sequences of simian immunodeficiency viruses from chimpanzees in Cameroon are strongly related to those of human immunodeficiency virus group N from the same geographic area. J. Virol. 74, 529–534 08 Martin, J. et al. (1999) Interclass transmission and phylectic host tracking in murine leukemia virus-related retroviruses. J. Virol. 73, 2442–2449 09 Lieber, M.M. et al. (1975) Isolation from the asian mouse Mus caroli of an endogenous type C virus related to infectious primate type C viruses. Proc. Natl. Acad. Sci. U. S. A. 72, 2315–2319 10 Todaro, G.J. et al. (1975) Infectious primate type C virus group: evidence for an origin from an endogenous virus of the rodent, Mus caroli. Bibl. Haematol. 115–120
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11 Fields, B.N. et al. (1996) Virology, Lippincott–Raven 12 Mettenleiter, T.C. (1991) Molecular biology of pseudorabies (Aujeszky’s disease) virus. Comp. Immunol. Microbiol. Infect. Dis. 14, 151–163 13 Tucker, A.W. et al. (1999) Evaluation of porcine cytomegalovirus as a potential zoonotic agent in xenotransplantation. Transplant. Proc. 31, 915 14 Ehlers, B. et al. (1999) Detection of two novel porcine herpesviruses with high similarity to gammaherpesviruses. J. Gen. Virol. 80, 971–978 15 Young, J.C. et al. (1998) New World hantaviruses. Br. Med. Bull. 54, 659–673 16 Selvey, L.A. et al. (1995) Infection of humans and horses by a newly described morbillivirus. Med. J. Aust. 162, 642–645 17 Halpin, K. et al. (1999) Newly discovered viruses of flying foxes. Vet. Microbiol. 68, 83–87 18 Chua, K.B. et al. (1999) Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 354, 1257–1259 19 Vitek, C.R. et al. (1996) Evidence against person-to-person transmission of hantavirus to health care workers. Clin. Infect. Dis. 22, 824–826 20 Wells, R.M. et al. (1997) Hantavirus transmission in the United States. Emerg. Infect. Dis. 3, 361–365 21 Stoye, J.P. et al. (1998) Endogenous retroviruses: a potential problem for xenotransplantation? Ann. New York Acad. Sci. 862, 67–74 22 Takeuchi, Y. et al. (1998) Host range and interference studies of three classes of pig endogenous retrovirus. J. Virol. 72, 9986–9991 23 Wilson, C.A. et al. (2000) Extended analysis of the in vitro tropism of porcine endogenous retrovirus. J. Virol. 74, 49–56 24 Paradis, K. et al. (1999) Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 285, 1236–1241 25 Morita, H. et al. (1998) A strategy for organ allografts without using immunosuppressants or irradiation. Proc. Natl. Acad. Sci. U. S. A. 95, 6947–6952 26 Starzl, T.E. et al. (1993) Chimerism after liver transplantation for type IV glycogen storage disease and type 1 Gaucher’s disease. New Engl. J. Med. 328, 745–749 27 Shustik, C. et al. (1995) A solitary plasmacytoma of donor origin arising 14 years after kidney allotransplantation. Br. J. Haematol. 91, 167–168 28 Breun, S. et al. (1999) Protection of MLV-vectors from human complement. Biochem. Biophys. Res. Commun. 264, 1–5 29 Leib-Mösch, C. et al. (1990) Endogenous retroviral elements in human DNA. Cancer Res. 50, 5636s–5642s 30 Pathak, V.K. and Hu, W-S. (1997) ‘Might as well jump!’ template switching by retroviral reverse transcriptase, defective genome formation and recombination. Semin. Virol. 8, 141–150 31 Zhang, J. and Temin, H.M. (1993) Rate and mechanism of non-homologous recombination during a single cycle of retroviral replication. Science 259, 234–238 32 Löwer, R. (1999) The pathogenic potential of endogenous retroviruses: facts and fantasies. Trends Microbiol. 7, 350–356
33 Gardner, M.B. (1993) Genetic control of retroviral disease in aging wild mice. Genetica 91, 199–209 34 Courgnaud, V. et al. (1992) Genetic differences accounting for evolution and pathogenicity of simian immunodeficiency virus from a sooty mangaby monkey after cross-species transmission to a pig-tailed macaque. J. Virol. 66, 414–419 35 Dorig, R.E. et al. (1993) The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 75, 295–305 36 Naniche, D. et al. (1993) Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J. Virol. 67, 6025–6032 37 Ward, T. et al. (1998) Role for beta2-microglobulin in echovirus infection of rhabdomyosarcoma cells. J. Virol. 72, 5360–5365 38 Bergelson, J.M. et al. (1995) Coxsackievirus B3 adapted to growth in RD cells binds to decay-accelerating factor (CD55). J. Virol. 69, 1903–1906 39 Donahue, R.E. et al. (1992) Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J. Exp. Med. 176, 1125–1135 40 Zhang, L. et al. (1999) Rapid clearance of simian immunodeficiency virus particles from plasma of rhesus macaques. J. Virol. 73, 855–860 41 Cornetta, K. et al. (1990) Amphotropic murine leukemia retrovirus is not an acute pathogen for primates. Hum. Gene Ther. 1, 15–30 42 Cornetta, K. et al. (1991) No retroviremia or pathology in long-term follow-up of monkeys exposed to a murine amphotropic retrovirus. Hum. Gene Ther. 2, 215–219 43 Kazazian, H.H. and Moran, J.V. (1998) The impact of L1 retrotransposons on the human genome. Nat. Genet. 19, 19–24 44 Eyre-Walker, A. and Keightley, P.D. (1999) High genomic deleterious mutation rates in hominids. Nature 97, 344–347 45 Peterman, T.A. et al. (1988) Risk of human immunodeficiency virus transmission from heterosexual adults with transfusion-associated infections. J. Am. Med. Assoc. 259, 55–58 46 Parrish, C.R. (1999) Host range relationships and the evolution of canine parvovirus. Vet. Microbiol. 69, 29–40 47 Klein, D. et al. (1999) Proviral load determination of different FIV isolates using Real-Time PCR: influence of mismatches on quantification. Electrophoresis 20, 291–299 48 Klein, D. et al. (2000) Accurate estimation of transduction efficiency necessitates a multiplex Real-Time PCR. Gene Ther. 7, 458–463 49 Takeuchi, Y. et al. (1996) Sensitization of cells and retroviruses to human serum by (alpha l-3) galactosyltransferase. Nature 379, 85–88 50 Rother, R.P. et al. (1995) Protection of retroviral vector particles in human blood through complement inhibition. Hum. Gene Ther. 6, 429–435 51 Berkowitz, R. et al. (1996) RNA packaging. Curr. Top. Microbiol. Immunol. 214, 177–218 52 Patience, C. et al. (1998) Packaging of endogenous retroviral sequences in retroviral vectors produced by murine and human packaging cells. J. Virol. 72, 2671–2676
Focus on infectious disease Molecular Medicine Today publishes a wide variety of articles on the molecular basis of infectious disease. Here’s a collection of recent articles to whet your appetite… Fraser, J. et al. (2000) Superantigens – powerful modifiers of the immune system. Mol. Med. Today 6, 125–132 Joo, M. and Hahn, Y.S. (2000) Animal models for immune defects caused by hepatitis C virus. Mol. Med. Today 6, 176–177 Dorrell, S. (2000) How HIV hijacks host cells. Mol. Med. Today 6, 140 Hewson, R. (2000) Hepatitis B transmitted via urine? Mol. Med. Today 6, 141 Future articles include: Expression library immunization for the systematic discovery of vaccines. (Sykes, K.F. and Johnston, S.A.) The role of nonhuman primate models of AIDS in vaccine development. (Johnston, M.I.)
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Xenotransplantation: is the risk of viral infection as great as we thought? Walter H. Günzburg and Brian Salmons
Two major hurdles remain before xenotransplantation can enter the clinic. The first is the more technical issue of being able to overcome the human immune response that leads to rejection of transplanted organs/cells from other species. The second, reviewed here, concerns the potential risk of inadvertent transfer of animal viruses present in the xenotransplant that are able to infect the human recipient. The threat from viruses is a particularly contentious topic because it poses a risk not only to those individuals who receive xenotransplants, but also to healthy individuals who come into contact, either directly or indirectly, with the xenotransplant recipient. In this review, we describe some of the virus types, in addition to the much discussed porcine endogenous retroviruses that might cross the species barrier, and assess the risk of such viruses causing disease in human hosts. THE rationale for investigating the feasibility of xenotransplantation includes: (1) the shortage of organ donors; (2) the avoidance of disease; and (3) the possibility of genetically engineering transplanted material. However, a number of hurdles, including: (1) the immune response to the xenograft; (2) differences in physiology between the donor xenograft and the recipient; (3) the production of suitable organs for xenotransplantation; and (4) the risk of infection to the patient and to society stand in the way of the routine application of xenotransplantation.
The infection risk in conventional allotransplantation Conventional allotransplantation has always been associated with the risk of transferring potentially infected material from the donor to the Walter H. Günzburg* PhD Professor and Chairman Institute of Virology, University of Veterinary Sciences, Veterinärplatz 1, A-1210 Vienna, Austria. Tel: 143 1 25077 2301 Fax: 143 1 25077 2390 *e-mail: [email protected] Brian Salmons PhD Scientific Director Bavarian Nordic Research Institute, Fraunhoferstr. 18b, D-82152 Martinsried, Germany.
transplant recipient. Although it is currently possible to screen for a wide variety of known pathogens, the risk remains, and both known and unknown microorganisms have been, and are still, unwittingly transferred. Obvious examples of known agents that are transferred to allotransplant recipients include the hepatitis viruses and HIV. A number of circumstances favour the risk of infection during allotransplantation. These include the urgency with which organs and tissues must be transplanted, often from donors who have only very recently, and without sufficient time for a thorough investigation, entered that status. Vascularization of the graft and direct cell–cell contact also contributes to the risk of transmission of infectious agents. Additionally, the heavy immunosuppression required for allotransplantation favours the establishment of many infections that would normally be easily dealt with by the immune system of the patient.
The infection risk in xenotransplantation Many of the problems associated with allotransplantation should be solved by xenotransplantation because standardized, well-characterized donor organs or tissues will be available ‘off the shelf’. However, a new problem of xenozoonosis might arise that, in contrast to the infections outlined above (which directly affect only the transplanted individual and close partners) could affect the patient’s family, friends, healthcare staff and, ultimately, the whole of society. It is because of this potential danger that the risk of xenozoonosis represents one of the most debated aspects of xenotransplantation. Only viruses will be considered in this article, because they represent a huge reservoir of potentially untreatable infections. Most known viruses can be excluded using a well-controlled specific pathogen free (SPF) environment. However, a few of the known viruses – specifically, those that integrate into the genome of the infected cell and do not
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lead to lysis of the cell – as well as the incalculable number of unknown viruses, pose a major threat.
Xenotransplantation compromises anti-virus defence systems The process of genetically engineering xenotransplantation donors (mainly pigs) so that their organs will not immediately be rejected upon transplantation into humans, concomitantly compromises the first line of defence of the human recipients against cross-species virus attack. Under normal circumstances, enveloped viruses, even if they are present in xenotransplanted material, would be rapidly eliminated by complement-mediated lysis (Box 1). One current goal, to prevent the rapid destruction of xenotransplants, is the inhibition of the complement system, for instance, by expressing complement regulatory proteins in the xenograft (Box 1). However, these regulators will also inactivate the highly effective virolysis system – which will no longer provide adequate protection against zoonotic viral infection (Fig. 1). An additional problem is raised by the circumvention of the physical barriers normally restricting entry. Usually, viruses must gain entry to a new host by overcoming physical barriers, such as the mucosal surfaces or the skin, without losing their infectivity. In the case of xenotransplantation, however, these barriers are effectively overcome by the direct implantation of potentially virus-harbouring material in the human, in such a way that it will be directly bathed by blood and other body fluids. The combination of SPF breeding conditions and rigorous testing guarantees that most of the known viruses are eliminated from organs before xenotransplantation. In these cases, the loss of the virolysis system and the lack of physical barriers would just mean the loss of a
Box 1. Control of hyperacute rejection of non-primate organs Non-primates, such as pigs, possess the enzyme a-1,3-galactosyl transferase (a-gal), and many of their structural proteins carry a-gal modifications. By contrast, humans and old-world primates do not express agal, but have natural antibodies (XNAs) directed against normal gut flora that cross-react with such a-gal modifications present on non-primate proteins. Thus, normal non-primate organs used for transplantation display a-gal residues on their surface, which, upon transfer into human recipients, are recognized by XNAs – triggering the activation of the complement system and leading to the hyperacute rejection reaction. A number of strategies have been used to eliminate the hyperacute reaction against transplanted non-primate organs. Circulating XNAs can be removed from the blood of a xenotransplant recipient. Alternatively, complement can be inhibited using monoclonal antibodies. Others have attempted elective depletion of complement by substances such as cobra venom factor, which can substitute for one of the members of the complement pathway but is not subject to feedback regulation, resulting in complement depletion. A number of regulators of complement are also known: they include; decay accelerating factor (DAF); membrane cofactor (MCP or CD46); factor H; and CD59. One of the most popular strategies involves using transgenesis to engineer the expression of one or more of these regulators on the surface of organs to be transplanted and thus protect them from complement-mediated lysis.
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backup safety system. However, there are circumstances in which the lack of these two natural safety systems might have grave consequences. The most serious of these are the persistent viruses (which either integrate or episomally maintain their genetic information without necessarily lysing the host cell), unknown viruses or those that cannot be detected using current test methods (and thus present a potential, unpredictable risk in xenotransplantation).
Persistent virus infection Persistent infections can be defined as those in which virus remains in specific cells rather than being cleared by the immune response. Indeed, immunosurveillance is often responsible for the maintenance of the persistent state. Well-known examples include cytomegalovirus (CMV) or Epstein–Barr virus (EBV); infection lies undetected until immunosurveillance breaks down as a result of immunosuppression – for example, during transplantation or during the later stages of AIDS – whereupon these viruses become clinically relevant. Other persistent infections are characterized by a progressive disease; well-known examples include HIV, its animal forms (SIV, FIV etc.) and other animal retroviruses, such as equine infectious anaemia1 and visna2 virus. However, non-retroviruses such as the measles virus can also potentially cause persistent infections, such as subacute sclerosing panencephalitis3. Chronic infections are those in which a balance has been attained. Either the rate of viral replication and cell destruction equals the production of new uninfected cells, or all cells are infected but there is no cell killing, so both viral- and cell-replication occur at the same time. Many persistent viruses escape elimination by the host immunemediated defence mechanisms by modulating or even evading the host immune system. Measles virus, for instance, restricts the expression of viral antigens in subacute sclerosing panencephalitis; HIV shows extreme antigenic variation in AIDS; CMV and adenoviruses both downregulate the expression of MHC class II; and CMV also alters the production of lymphocyte adhesion (ICAM-1, LFA-1) or co-stimulatory (B7) molecules. Another strategy followed by viruses such as HIV, herpesviruses (such as EBV) and poxviruses is to modulate the production of cytokines by the cell, and many viruses (for example, influenza) have specific strategies to downregulate the production of the classical antiviral, interferon. In all these scenarios, a change of host can upset the delicate balance between virus and cell replication, or change the pathogenicity profile of the virus. Prolonged incubation periods might, however, remain a feature of these infections, making early detection difficult. It is not clear whether the immune modulatory strategies practised by many viruses will still be active after trans-species transmission of a virus, or whether, with time, there will be a breakthrough of a frank viral disease.
Persistent retroviral infections Probably the most important, and certainly the best-understood, category of persistent viruses are the retroviruses. These enveloped viruses have an RNA genome that is reverse transcribed into a doublestranded DNA ‘proviral’ form after infection of the target cell. The proviral DNA is integrated in a random fashion into the host-cell DNA and persists for the life span of the cell. For instance, HIV persists in infected individuals despite aggressive highly active anti-retroviral chemotherapy (HAART) regimes (also known as triple therapy), because of the proviral state. If a retrovirus infects a host germ-cell, the proviral information becomes part of the genetic information of the offspring, and is termed an endogenous retrovirus. Thus, persistence of
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Figure 1. Xenotransplantation eliminates the antiviral defence system. (a) Enveloped viruses, such as retroviruses, arising from non-primate cells incorporate part of the plasma membrane of the cell from which they bud. Consequently, they display proteins bearing the a-1,3 galactosyl transferase (a-gal) modification on the surface of virions, and are recognized by xenoreactive natural antibodies (XNAs) present in human serum, triggering complement-mediated lysis or virolysis. This figure shows the viral RNA being packaged by viral core proteins and budding from the producer cell. Budding involves the core moving through the plasma membrane and becoming coated with this membrane. Viral surface-proteins present in the membrane, as well as a-gal modifications, will therefore be displayed on the surface of the viral particle. XNAs generated against crossreacting human gut-flora react with the a-gal modifications and trigger complement mediated lysis (virolysis) of the virus particle49. (b) Enveloped viruses, such as retroviruses, arising from transgenic xeno-organs engineered to express complement regulators (to prevent hyperacute rejection) will also incorporate these complement regulators when they bud through the plasma membrane. Expression of these complement regulatory proteins on the surface of the budding virion allows it to escape virolysis28,50.
viral genetic information is not restricted to the infected animal but is found in every successive generation. Like any other cellular gene, the integrated provirus is transcribed by the cellular machinery, and the resultant RNA used both for translation into viral structural proteins and as a genome for the resultant retroviral progeny4. Retroviruses are associated with tumour induction, immunodeficiencies and neurodegenerative diseases although often only by an indirect mechanism4. Retroviruses have, in the past, been known to jump species. It is now generally accepted that SIV jumped from infected monkeys to humans giving rise to HIV in the 1930s5–7. Because of the endogenous nature of other retroviruses, it is also possible to analyse the sequence relatedness of these ancient proviruses. Recently, the sequences of endogenous retroviruses related to murine leukaemia virus (MLV) have been compared in a number of different species. Phylogenetic analysis revealed that viruses isolated from a particular host generally cluster together, suggesting that horizontal transmission of infectious virus between species rarely occurs. Nevertheless, evidence was found for two instances of cross-species transmission; one from mammals to birds, which was followed by rapid spread to other avian hosts; the other involved the transmission of a gibbon ape leukaemia (GaLV) retrovirus to koala bears, possibly via rodents8, although previously it has been speculated that this virus was originally transmitted from mice to gib-
bons9,10. Disturbingly, both GaLV and the koala virus are closely related to porcine endogenous retroviruses (PERVs; see below)8.
Persistent herpesvirus infections The persistence of a virus can also result from the establishment of a latent infection, as in the case of herpesvirus infection, in which virus cannot be detected between episodes of recurrent disease. These viruses are extremely problematic, even in conventional allotransplantation. Herpesviruses are enveloped viruses with a DNA genome that can be maintained latently (in other words, non-expressed) for the life of the host cell as a circular episomal form. The latent form of the virus is often transmitted from the donor to the organ/tissue recipient. Not only might such latent herpesvirus infections persist undetected for long periods before being activated by stress or environmental factors, but reactivated herpesviruses, which cause death of the host cell during their lytic cycle, also frequently show an increased pathogenic profile after cross-species transfer, leading to highly virulent forms of disease11. As pigs are the most likely potential xenograft donors, the search for potentially zoonotic herpesviruses is on. Until recently, only two herpesviruses that infect pigs were known: pseudorabies virus, an aherpesvirus, which causes encephalomyelitis and respiratory tract inflammation12; and porcine cytomegalovirus, a b-herpesvirus, found in 201
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the respiratory tract of pigs, which causes atrophic rhinitis13. Recently, however, two g-herpesviruses have been discovered (porcine lymphotropic herpesvirus types 1 and 2)14 and it seems that other herpesviruses that have yet to be detected might be present in some pigs.
Other persistent viral infections Besides these viruses, there are other viruses that show abnormally long time lags between the infection event and viral replication and spread through the body. One such well known virus is the rabies rhabdovirus11, and other as yet unknown members of the rhabdovirus family might show similar biological properties. Other animal viruses, the human counterparts of which have the potential to cause debilitating chronic infections, such as hepatitis (members of a number of virus families) or neoplasia (papilloma or polyoma viruses) are also of concern. Although it would be possible to screen for known persistent viruses in organ donors (Box 2), as yet unknown viruses probably also exist. The most dangerous of these are viruses that have co-evolved with their host – so there is an undetected or chronic infection that does not produce disease in the natural host. However, potentially long latency periods after transfer to the human recipient, coupled with possibly altered pathogenicity profiles, mean that these viruses, and the resulting illnesses, can only be determined after the event. Time and time again, new examples arise of viruses that apparently have existed in perfect symbiosis with their natural animal host for many years, and thus have not been recognized owing to their lack of pathogenic phenotype, but that cause human disease upon cross-species transmission. Examples of such viruses classically include Ebola and Marburg virus disease, SIV/HIV, and most recently Hanta, Hendra and Nipah viruses. Interestingly, these viruses are all enveloped viruses transmitted from animals such as non-human primates (Ebola, Marburg, SIV/HIV), rodents (the Sin Nombre Hanta virus15), bats (Hendra16,17) and pigs (Nipah18), although fruit-eating bats are probably a reservoir of Nipah virus. In these cases, even with an intact virus defence system, these zoonotic enveloped pathogens were quickly able to establish themselves in humans and manifest their pathogenicity. At least for some of these viruses, subsequent human-to-human spread has
Box 2. Molecular methods for screening for new zoonotic agents A number of molecular techniques are now available for the very sensitive detection of viral genetic information. They are usually based on a polymerase chain reaction (PCR) and include conventional DNA- and reverse transcriptase-PCR, nested PCR and more sophisticated PCRbased methods such as Taqman Real-Time PCR and in situ PCR. However, these techniques are sensitive to small variations in genome sequence – raising the possibility of false negatives47. An additional problem, not often discussed, but highly relevant to the prevention of false negatives, is the correct and reproducible pre-assay sample preparation and nucleic acid extraction; multiplex PCR techniques have been developed to address this48. Thorough validation regimes are required to ensure the quality of the PCR results obtained. Unknown viruses can only be traced by their similarity at the nucleic acid level to other viruses in their family, using redundant PCR approaches. This puts severe constraints on the detection systems because the degree of nucleic acid homology between individual viruses of the same family can vary considerably.
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been documented (Ebola, HIV). However, it appears that this has only been observed after initial zoonotic transfer from non-human primates. There is no evidence for human-to-human spread of zoonotic viruses of non-primate origin, although this has been extensively studied for the rodent-originating Sin Nombre virus19,20. All these viruses were unknown before the outbreaks of disease they caused in humans. It would be unwise to assume that there are no more of these human pathogenic viruses lurking in the animal world.
Pigs as potential organ donors Both physiological and ethical reasons suggest that the majority of, if not all, xenotransplants will be obtained from pigs. Non-human primates could represent a source of organs for babies and very young children but, in these circumstances, it might even turn out that the immunological problems facing these special situations can be overcome without resorting to transgenesis. A whole range of potentially zoonotic porcine viruses are known (Table 1). Also, viruses that are normally not regarded as zoonotic but that can replicate in human cells under laboratory conditions, as well as those that could recombine to form a potentially new type of virus, must be considered.
Porcine endogenous retroviruses Of all the potential porcine infectious agents, the porcine endogenous retroviruses (PERVs) have aroused the greatest interest. Assays for the presence of PERV DNA will always yield positive results because all examined pigs contain a number of these viruses integrated into their genomes. So far, PERV-A, PERV-B and PERV-C have been characterized. Analysis of pigs worldwide indicates that they contain 10–25 copies of PERV-A, around ten copies of PERV-B and either none or a variable number of copies of PERV-C at distinct integration loci21. There is no information currently available as to which of these loci produce infectious virus or potentially pathogenic viral proteins. PERV-C appears to be severely compromised in its ability to infect human cells, whereas relatively good infection titres have been obtained with PERV-A and -B (Ref. 22). Further passage of the virus produced from such infected human cells leads to increased infectivity of human cells in vitro23.
Spread of PERVs to humans What is the chance of these or other porcine viruses spreading to human recipients after xenotransplantation? In recent studies24, 160 patients who had been exposed to living porcine cells in the course of various treatment regimes for periods of up to 460 days were analysed. All patients tested negative for porcine virus transmission except for some of the patients who received extracorporeal splenic perfusion in Russia. These patients were in contact with porcine cells for 50–60 min. Of the 30 positive samples from the Russian group, 23 showed clear evidence of circulating pig DNA as determined by the presence of other porcine nucleic acids of mitochondrial or centromeric origin. This suggests the presence of porcine cells in these patients (chimerism) and, thus, it is possible that the positive PERV DNA signal was derived from integrated copies in these porcine cells, rather than from infection events. Not enough DNA was isolated to permit the analysis of the remaining seven samples. The chimerism suggested by the presence of mitochondrial or centromeric porcine DNA (microchimerism) was observed for up to 102 months. Others have described microchimerism specifically after spleen-cell transplantation into non-immunosuppressed and non-irradiated mice, which leads to lasting tolerance25, but for shorter periods. Paradis and
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Table 1. Some zoonotic and potentially zoonotic porcine viruses Transmission/ infection of human cells
Virus family
Virus name
Porcine disease
Remarks
Zoonotic
Orthomyxoviridae
Influenza
Influenza
Zoonotic
Paramyxoviridae
Nipah
Zoonotic
Flaviviridae
Japanese encephalitis (JE)
Zoonotic
Rhabdoviridae
VSV
Vesicular stomatitis
Zoonotic
Picornaviridae
Aptho
Foot and mouth disease
Zoonotic
Picornaviridae
Entero
Swine vesicular disease
Zoonotic Zoonotic Probably zoonotic
Rhabdovirus Poxviridae Parvoviridae
Lyssa Vaccinia Parvo (PPV)
Rabies Swinepox-like SMEDI-syndrome
Probably zoonotic
Herpesviridae
Aujeszky’s disease
Possibly zoonotic Possibly zoonotic
Poxviridae Picornaviridae
Pseudorabies (PRV) Suipox Cardio
Killed 20–40 million people (1918–1919; Spanish influenza). Possesses a segmented genome – virus contains eight pieces of RNA, each encoding a different gene. Reassortment results in new variants with new properties, e.g. reassortment between pathogenic porcine viruses and highly transmissible human viruses formed the H1N1 strain, which is lethal to humans Newly described in Malaysia: has killed over 60 adults. First described in workers exposed to pigs Fatal human disease transmitted by mosquitoes, with virus amplification in pigs and birds. JE virus exists in Asia, where swine raising is common. Infections in pigs are generally inapparent, except for stillbirth or abortion in pregnant swine, and aspermia in boars Found in many mammals, probably transmitted by mosquitoes and sandflies. Infected humans show transient symptoms of influenza Very few cases, disease is relatively benign in humans; subclinical or similar to animal symptoms Porcine variant of the human pathogen Coxsackievirus B, it causes a high degree of morbidity in infected animals and an influenza-like illness in humans Infects all warm-blooded animals and is almost invariably fatal Many original outbreaks of swinepox were caused by vaccinia A known contaminant of cell-culture trypsin and of blood products (Factor VIII). Can infect human cells, 50% of porcine islet recipients develop antibodies to PPV Wide host range, infects human cells in vitro. Recently, three patients seroconverted after previous contact with a cat that died of PRV Usually a mild disease with lesions restricted to the skin Wild rodents act as a reservoir for the virus. Tropism for the myocardium leads to rapid death in about 80% of infected cases Type 1 widely distributed in swine, but does not appear to be associated with any major disease symptoms. Type 2 has been identified as associated with post-weaning wasting disease syndrome in pigs. Antibodies to Type 1 have been identified in humans Widely distributed as endogenous viruses in the germline of pigs; originally isolated from a lymphosarcoma. Although expressed in a variety of porcine tissues, and capable of infecting human cells in vitro, there is no evidence for their replication and spread in humans Causes hepatitis and is closely related to human hepatitis E virus. Transmission to humans might be possible because the homologous human hepatitis E virus, which is associated with a 20% mortality rate in infected pregnant women, has been used to infect pigs Porcine polioencephalomyelitis Highly contagious viral disease of swine. The infection can run an acute (killing up to 90% pigs in a herd), subacute, chronic or inapparent course, mainly depending on the virulence of the virus. Antigenically related to bovine viral diarrhoea virus (BVDV) Transmissable gastronenteritis; vomiting and wasting disease; epidemic diarrhoea; respiratory disease Can infect swine PRRS causes abortion and premature birth as well as general fertility and respiratory problems
Swinepox Encephalomyocarditis
Potentially zoonotic Circoviridae
Circo
Post-weaning wasting syndrome
Potentially zoonotic Retroviridae
PERV
Lymphosarcoma?
Potentially zoonotic Floating genus
Hepatitis E
No evidence No evidence
Picornaviridae Flaviviridae
Entero Pesti
Teschen/Talfan disease Hog cholera
No evidence
Coronaviridae
Corona
Various
No evidence No evidence
Paramyxovirus Arteriviridae
Rinderpest Arteri
No evidence No evidence
Reoviridae Herpesviridae
Rota b herpes
No evidence
Asfarviridae
No evidence No evidence
Caliciviridae Flavivirus
African swine fever Vesi Pesti
Rinderpest Porcine respiratory and reproductive syndrome (PRRS) Diarrhoea Cytomegalovirus disease Widely distributed in swine and associated with inclusion body rhinitis and reproductive failure. No evidence that humans are infected, however, human CMV is a major problem in transplantation medicine African swine fever Limited to members of the Suidae – all attempts to infect members of other species have been unsuccessful Vesicular exanthema Eradicated Bovine viral diarrhoea Widely spread in cattle. The virus can also infect pigs, sheep and other ruminants leading to persistent lifelong infection and shedding
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Glossary Antigenic variation – Altered protein composition, which is recognized by the immune system. Antisense – Nucleic acid molecules generated by reversing the orientation of the transcribed region of a gene, which then forms a duplex with the natural ‘sense’ mRNA transcript, thereby preventing its translation. Autologous – Derived from the same individual. Chimerism – A state in which two or more genetically different populations of cells co-exist. Co-stimulatory molecules – Molecules that mediate interactions between immune cells. Cytokines – Intercellular signals, usually proteins, involved in the regulation of cellular proliferation and function. Interferon – Secreted molecule induced by virus replication that blocks protein synthesis and cell growth, and thus blocks viral multiplication. Lymphocyte adhesion molecules – A family of mammalian carbohydrate-binding adhesion molecules that mediate immune-cell interactions. MHC class II – A cluster of closely linked genetic loci encoding class II polypeptide products involved in the generation and regulation of immune responses. Provirus – A viral genome that is integrated into the genome of its host cell and transmitted to its progeny cells and, when integrated into the genome of a germ cell, from generation to generation. Pseudotyping – Pseudotyped or phenotypically mixed virions can arise when the same cell is simultaneously infected with two enveloped viruses. Such virions usually consist of the core and genome from one virus and the envelope from another. Reassortment – The process by which a novel virus arises from a cell that is co-infected with two viruses. It involves exchange of viral genetic information. Ribozymes – Catalytic RNA molecules that can promote specific biochemical reactions without the need for ancillary proteins – in this case the specific cleavage of viral RNA. Xenotransplantation – The transfer of organs or tissues from a donor animal of one species to the recipient of another species (e.g. pig to human). Xenozoonosis – A zoonotic infection that can be transmitted by the xenotransplantation of animal tissues or organs. Zoonotic infection – A disease that is transmissible from nonhuman vertebrates to humans.
colleagues speculate that the signal comes from porcine dendritic- or stem-cells that are only expressing low levels of a-l,3-galactosyl transferase (a-gal), thus allowing them to escape antibody-mediated clearance. Although it is known that human allotransplantation of solid 204
organs transfers passenger cells that might give rise to a state of persistent microchimerism26,27, this is after initial immunosuppression. Whether the observed PCR signals really originated from microchimerism, and whether sequencing would have provided more information, remain unanswered questions. In addition, this study did not reveal any evidence for pathogenicity mediated by other porcine viruses, even though some patients were followed up for periods of up to 102 months (eight and a half years). Although such events cannot be ruled out, they are probably very rare. However, neither of these findings are unexpected – what might happen when humans are exposed over long periods to modified porcine viruses that incorporate complement regulators that could disarm the human virolysis system28 remains unknown. Also, the study does not address issues relating to organ xenotransplantation, in contrast to islet and skin transplants or extracorporeal perfusion, where it is to be expected that both the time of exposure and the connection to the circulatory system will increase the amount of infectious virus that contacts the host.
Recombination of PERVs with human endogenous retroviruses It has been estimated that 1–5% of the human genome comprises retroviral or retrovirus-like sequences29. Most of these endogenous retroviruses represent partial retroviral genomes, often solitary long terminal repeats (LTRs) (Fig. 2), which themselves are not biologically active, although they can be transcribed into RNA. Although HERVs have not been shown to give rise to infectious retrovirus29, they do represent a potential source of material that could take part in recombination reactions with porcine retroviruses to create new, potentially pathogenic variants. Recombination in retroviruses is believed to occur mainly at the RNA level30 (Fig. 2) and is favoured by sequence homology. Recombination between non-homologous retroviral genomes is 100–1000-fold less likely than that between homologous ones31. Human and porcine retroviral sequences share little sequence homology8, thus the chance of recombinants arising is probably equal to that of recombination with any heterologous piece of RNA. By contrast, the chance of intra-species recombination of retroviral sequences to generate new viruses seems much higher.
The PERV-free pig: dream or reality? To eliminate the potential risks from PERVs that can infect human cells it will be important to remove those PERV loci that are biologically active from the porcine donors. If all the identified loci are biologically active, this would not be feasible. However, it seems more likely that only a few are biologically active, considering what is known about human32 and murine33 endogenous retroviruses; and if the active viruses could be identified it would be possible to inactivate them using current knockout technology. It will be difficult to conclusively evaluate which are biologically active, and it cannot be ruled out that the xenograft environment (for instance immunosuppression, or other drug treatments) might not activate a previously silent endogenous copy. It is therefore unrealistic to imagine that a PERV-free porcine organ donor can be produced in the foreseeable future. Nevertheless, a number of extra safety mechanisms, which are analogous to the safety devices used in gene therapy, could be built into the pig to ensure that, should one of the other PERVs become active for any reason, its expression could be controlled and eliminated. Such safety devices include the use of antisense technology to control PERV expression, ribozymes directed against PERV RNA, antivirals developed against points in the PERV life cycle and immunization to prevent infection.
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(a)
(j) (b)
R U5
RNA
R U5
Nucleus
(c) DNA
RNA
RNA
U3 R U5
(d) R U5
DNA
U3 R
(k)
U3 R U5
U3 R U5
gag
LTR
gag pol env
pol
env
LTR
U3 R U5
U3 R U5
RNA
U3 R
Reverse (l) transcription
gag pol env U3 R U5
U3 R
(m)
(e) R U5
U3 R
(f)
First host cell
(h)
(g)
(i)
Nucleus RNA
R U5
U3
R
DNA RNA
R U5
U3
U3 R U5
gag pol env U3 R U5
R
RNA
R U5
gag pol env U3
Vir particles contain two Virus different viral RNAs
Second host cell Molecular Medicine Today
Figure 2. Retroviral recombination. Retroviruses are the only viruses with a diploid genome. This means that two copies of the viral RNA genome are packaged into the virion. The RNA genomes are identified and packaged by virtue of specific sequences known as packaging signals51. However, even in the absence of these signals, heterologous RNAs can be packaged52. If two disparate molecules of RNA are packaged into a virion, recombination can occur during reverse transcription. It is believed that the reverse transcriptase enzyme responsible for generating the DNA copy, which will become integrated into the target cell as the provirus, can jump from one RNA molecule to the other, copying a part of one and then juxtaposing the copy of the other molecule. This template-switching is most probably favoured by template homologies, although it appears that it can also take place in its absence. (a) A retrovirus infects a host cell by virtue of an interaction with a specific receptor. (b) The retrovirus is internalized, uncoated and the viral RNA genome is reverse transcribed to a DNA form. (c) The DNA form moves to the nucleus and integrates in the genomic DNA of the target cell. (d) The integrated provirus is then transcribed to generate viral RNA, which is used to make viral structural proteins, and is packaged into newly formed virions as their genetic information. Other endogenous retroviral sequences also present in the cell might be transcribed into RNA (e). If these RNAs carry the appropriate packaging signals (f), they too can be packaged into newly formed virions. Even retroviral RNAs lacking the correct packaging signals and non-retroviral RNAs can be packaged at a low frequency. (g) The retrovirus particle buds from the cell. Retroviruses carry two copies of their genetic information, so they can package two different RNAs, as shown here (white and grey). (h) The virus particle infects a second host cell and is internalized. (i) Recombination can occur during reverse trancription (see inset): (j) Viral DNA synthesis is primed by a tRNA molecule bound to the (white) viral RNA. DNA synthesis proceeds to the end of the template RNA. (k) As a result of the terminal duplications of the R sequence in the viral RNA, the nascent DNA strand may become translocated to the R region on the second (grey) RNA template. During further DNA synthesis (l) a second-strand jump from the grey RNA to the white RNA (m) will result in the generation of a hybrid DNA molecule carrying part of the white and part of the grey coding sequences. After integration and transcription of this provirus, new virus particles will emerge carrying the genetic information of the hybrid virus.
Other types of porcine viral recombinants It has long been known that when a cell is infected with two different viruses, novel viruses can emerge with new properties. The new viruses can result from: (1) recombination at the genetic level; (2) mixing of viral proteins (pseudotyping); or (3) mixing of viral genetic information (reassortment). These novel viruses are unpredictable and can
gain a novel pathogenic phenotype in the host; for instance, reassortment in influenza can lead to antigenically-shifted variants that cause severe public health problems. If the event occurs at the genetic level (recombination or reassortment), the change will be fixed over the next generations, whereas even pseudotyping can open the possibility of entry into new types of cells with subsequent secondary effects. 205
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Adaptation of porcine viruses to the human environment
Once a virus that originates in a non-primate (and thus has the a-gal modification) has replicated in a human cell, the progeny virions will no longer carry the a-gal modification and, thus, will no longer be sensitive to human complement-mediated virolysis. This adaptation of the virus to its new host usually results in an acceleration of the speed of successful replication. Viruses in general, and especially RNA viruses, show a high rate of mutation. Enhanced replication increases the chance of mutational change, leading to greater diversity in a virus population. This might favour the production and selection of a virus capable of efficiently infecting human cells. For instance, alterations in pathogenicity after cross-species transmission of SIV have been associated with mutation34. Infection is dependent on the presence of the appropriate receptor molecule on target cells. Nevertheless, viruses can be coerced into using new receptors. Current thinking would dictate the construction of transgenic porcine donors that express complement regulatory molecules in an attempt to regulate the hyperacute rejection reaction (Box 1). However, complement regulators such as CD46 or CD55 are natural receptors for measles35,36, echo37 and coxsackie B (Ref. 38) viruses. It is therefore possible that porcine viruses might evolve to use these receptors in the donor pig; after xenotransplantation, these viruses would be free to use these natural human molecules as receptors to enhance their spread through the patient.
Xenotransplantation and immunosuppression Finally, it should not be forgotten that, initially, xenotransplantation will probably require a more intense immunosuppression regime than currently practised for allotransplantation. Immunosuppression will increase the likelihood of a xenoinfection, giving viruses a better chance to adapt to their new environment. Furthermore, immunosuppression can result in the activation of persistent viruses, such as EBV and CMV, and might allow retrovirally-induced diseases to develop. Indeed, this is critical in models of retrovirus-mediated pathogenesis where new-born animals are inoculated in order to avoid the suppressive immune response.
Virological risks arising from xenotransplantation The virological risks associated with xenotransplantation can be divided into those affecting the patient and those affecting the community. In the light of probable medical benefit to the patient, the first risk can be negated in terms of the risk–benefit pay-off. The risk to the community, however, is of some concern. If the pathogenic phenotype of the porcine (or porcine–human recombinant) virus manifests itself early after transplantation, it might be possible to detect it early enough by a sophisticated screening procedure (Box 2). However, viruses causing no immediate disease will be very difficult to detect. Similarly, if at a later time (perhaps years later), the xenotransplanted patient becomes infected with a human virus that crosses into the xenotransplant and recombines with the porcine virus to generate a novel pathogenic virus, it will be practically impossible to identify until it is too late. Such a rare event could only be detected, and possibly prevented from spreading, by a life-long health status monitoring programme, and it will be extremely difficult to legislate for this remote possibility.
Risk assessment Can we put a figure on the likelihood that such events occur? This is, of course, very difficult. However, we can make some rough calculations that might help us to understand the potential size of the prob206
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lem. The number of known viruses that are zoonotic is small, thus the chance of an unknown virus proving to be zoonotic and readily transmitted in the human population is also small. Nevertheless, viruses, especially those with an RNA genome, can rapidly adjust to new environments, making the chance of adaptation, especially in the face of immunosuppressive regimes and the use of new receptors, quite likely. On the other hand, the virus that emerges will not necessarily be able to spread in the human population. Even if a virus adapts to the human cellular environment, can replicate there, and be transmitted from the xenotransplant recipient to the human population, it will rapidly elicit a controlling immune response.
Risk to the patient In one study, in which cells producing replication-competent MLV were infused into immunosuppressed non-human primates, lymphomas and viraemia were observed in three of 12 animals, and the animals died after around 200 days39. In these experiments, around 108 cells, titred in cell culture as producing at least 103 replication competent virus particles ml21, were introduced. Assuming that the in vitro titre was calculated from 106 producer-cells over a 24 h period, and assuming an in vivo virus half-life of around 4 min (as recently calculated40), the steadystate level of circulating infective virus particles should have been around 103 at any given time. However, titres of 104–105 infectious particles ml21 were measured in sera from animals developing lymphoma at the time of death. Since the plasma volume of the macaques used in these studies is calculated at about 30 ml kg21 (Ref. 40), and assuming the animals weighed around 10 kg, this would mean a total amount of infective virus of the order of about 107. Thus, in the three animals that developed lymphomas, the virus must have infected additional target cells, replicated and been released from these cells, thereby increasing the viral titre exponentially (<14 doublings of titre must have occurred over the 200-day period, compatible with an average doubling of titre every two weeks). Interestingly, all ten primates were subject to highdose total-body irradiation, and yet only three of the animals were unable to control the replication-competent virus infection. Seven of thes e animals were examined further. Of the five that controlled the infection, all showed serum antibody that was reactive against viral protein, whereas the two animals that developed lymphoma apparently failed to mount a humoral response to the virus. The finding that antibodies are produced after total-body irradiation seems surprising, but suggests that antibody production is possible even after severe immunosuppression. The data underline the importance of a humoral response in controlling infection in the autologous setting. Virus being produced from such autologously transplanted cells will not be subject to a-gal-triggered, complement-mediated virolysis. Thus, we might expect that virus arising from surface-modified transgenic xeno-organs should, in most cases, be controlled by a rapid antibody response, even in the immunosuppressed recipient. Given the more controllable nature of the xenotransplantation process, it might even be feasible to vaccinate recipients against known viruses, such as PERVs, before transplantation to ensure a good humoral anti-viral response. The data presented by Donahue and co-workers39 demonstrates, however, that replication-competent viruses can cause tumours in 30% of infected macaques under immunosuppressive conditions. It is not clear whether such viruses will be pathogenic in humans, indeed, other studies using both immunosuppressed and immunocompetent primates found no evidence of pathogenicity41,42. It is clear from human retroviral infections that there is no evidence for a common integration-site tumorigenic mechanism in patients infected with HIV-1, HIV-2, human T-cell
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The outstanding questions Are PERVs associated with disease in humans? • Do porcine cells harbour as yet unidentified zoonotic • viruses? porcine zoonotic viruses be transmitted from human• Will to-human? incorporation of immunomodulatory molecules en• Will hance viral pathogenicity? long will patients have to be monitored after xeno• How transplantation?
leukaemia virus-1 (HTLV-1) or HLTV-2. This is surprising, particularly for HIV-1, in light of the large number of patients infected, as well as recent calculations showing that 1012 virus particles per day are produced by such patients40. Thus, it could be argued that human retroviral infections contrast starkly with those of the animal world – where tumorigenesis by insertional mutagenesis is the rule, rather than the exception – a difference that might reflect the higher stability of the human genome. Indeed, the frequency of retrotransposon-induced disease-causing mutation in the human population has been estimated at 1:670, but none of these were the result of retroviral insertion43. From this point of view, it could be argued that PERV infection, even in the case of a productive infection, will not represent a great danger to the individual patient.
Risk to the germline Even the risk to the germline might be overstated in these patients. Although the use of condoms could protect against the spread of infectious agents, it has been argued that the prevention of procreation by xenotransplant recipients should also protect against the effects of harmful retrovirus-induced mutations in the human genome. However, it has been demonstrated that the human genome per se underlies an extremely high deleterious mutation rate – and yet the human population survives43,44. It is extremely unlikely that a rare integration into one germline cell will have a large influence on this.
or viruses that only cause symptoms after very long periods, could easily spread without being noticed. This can occur rapidly as has been documented for a feline parvovirus spreading to the dog population worldwide within a matter of years46.
Concluding remarks In conclusion, there is a certain level of risk of viral infection associated with xenotransplantation, and caution is warranted to prevent the emergence of new diseases, but it is difficult to quantitate these risks. In view of the enormous potential benefits associated with xenotransplantation, and the fact that no further progress can be made in the absence of further experimentation, it would be unwise to discontinue work in this area. The most probable candidates for viruses that could be a danger to society are those that show good infectivity and are difficult to contain, namely the persistent viruses with a long latency period. Thus, it is imperative that xenotransplanted patients are centrally registered and closely followed for health- and virus-status to detect emerging viruses over extended periods (Box 2). A battery of screening tests, based on our considerable knowledge of potentially zoonotic agents, as well as of physiological changes associated with viral infections in general, are essential to minimize potential risks to society, at least at present. Although this will not guarantee the detection and prevention of a new pathogenic virus, and although scenarios exist whereby the recombination might take place very late – even years – after transplantation, the health and virus screening programme should be conducted at least for the first five years after xenotransplantation. It will, however, be safe to assume that when xenotransplantation has become sufficiently established that patients survive for an average of five or more years, and when a sufficient number of patients (about 200) have been subjected to such a five-year screening programme without any signs of pathogenic virus release, the requirement for such post-operative screening could be readdressed. Acknowledgments. WHG would like to thank the Xenotransplantation Project Group of the Europäische Akademie Bad Neuenahr-Ahrweiler, Germany and their guests for stimulating discussions and support. The authors also gratefully acknowledge the constructive criticisms and suggestions of the reviewers.
Risks to society
References
What are the risks to the population at large? Uncontrolled virus production presents a theoretical risk to the entire human population – particularly to those in close contact with xenotransplant recipients. However, it should be remembered that retroviruses are, by nature, labile and are not transferred by aerosol (unlike other classes of virus that might play a role in xenotransplantation, such as influenza). They are susceptible to dehydration, can only be passed in body fluids and, even then, they are not very contagious45. Therefore, like HIV, their spread could be limited by health-control measures. More dangerous are novel aerosol-borne recombinants or human adapted viruses. Even in these situations, it should be possible to contain the virus quickly, provided symptoms develop rapidly. Recent scenarios in which rapidly fatal viruses, including Hendra, Nipah and Ebola, have been transmitted from animal hosts to humans reveal that, once recognized, spread of infection can be limited very quickly. Although it is conceivable that, with modern air transportation, such a viral disease might be transmitted to a large number of people before the symptoms are identified and the patient isolated, the speed of the outbreak of symptoms makes this unlikely, albeit not impossible. By contrast, non-pathogenic or weakly-pathogenic viruses,
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Focus on infectious disease Molecular Medicine Today publishes a wide variety of articles on the molecular basis of infectious disease. Here’s a collection of recent articles to whet your appetite… Fraser, J. et al. (2000) Superantigens – powerful modifiers of the immune system. Mol. Med. Today 6, 125–132 Joo, M. and Hahn, Y.S. (2000) Animal models for immune defects caused by hepatitis C virus. Mol. Med. Today 6, 176–177 Dorrell, S. (2000) How HIV hijacks host cells. Mol. Med. Today 6, 140 Hewson, R. (2000) Hepatitis B transmitted via urine? Mol. Med. Today 6, 141 Future articles include: Expression library immunization for the systematic discovery of vaccines. (Sykes, K.F. and Johnston, S.A.) The role of nonhuman primate models of AIDS in vaccine development. (Johnston, M.I.)
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