4th National Symposium on Biosafety

Xenosis from Swine: Assessing the Infectious Risks of Xenotransplantation

Jay A. Fishman, MD
Associate Chief and Clinical Director
Transplantation Infectious Disease Unit
Massachusetts General Hospital and Harvard Medical School
Charlestown, MA 02129
617-726-5772

The main limitations of organ transplantation have been the need for immune suppression to maintain graft function and the resultant incidence of opportunistic infection observed in these individuals. Dr. Jonathan Allan has talked extensively about the potential risk of using non-human primates as donors. In part, one of the reasons for concern about the use of non-human primates as doors for human transplantation is because our knowledge base is greater and thus the defined risks are possible larger. This is due, in large part , to the AIDS epidemic and the use of non-human primates as models for AIDS and as models for retroviral infection. Thus, no realistic comparison between potential donor or source species for xenotransplantation can be made until the knowledge base for swine approaches that of primates. There is, however, the theoretical disadvantage of nonhuman primate organ donors because of the very genetic relatedness that decreases the level of immune suppression expected to be needed to maintain xenograft function. This reflects the likelihood that receptors and intracellular transcription factors (the intracellular environment) of primate donors may be similar to that of humans and that viruses (in particular) may be more easily transferred between species in this setting.

The goal for all of us if xenotransplantation is going to be a viable clinical option, is to minimize the incidence and risk of opportunistic infection in the recipient and in the community at large. The mechanisms to achieve this goal involves a spectrum of research aimed at the determination of potential pathogens derived from other species in the setting of relevant forms of immune suppression, for various kinds of transplants. Basically, the assessment of risks in animal models.

The types of disease we're looking at can be categorized for ease of discussion. Some are very common, such as Toxoplasma gondii. These are zoonotic agents known to cause infections in humans. Some of them are species specific; cytomegalovirus, and most of the common herpes viruses are probably a good example of species specific microbes. Organisms of broad host range, such as reovirus an other viruses which tend to be able to infect a larger spectrum of hosts are likely to be a basis for concern. However, the main issue is the group of unknown and unidentified organisms, particularly the retroviruses which are of unknown quantity and importance in the future of transplantation. And what we end up with are four potential types of clinical syndromes from these infections:

The risk of infection is really a function of the level of immune suppression that is necessary to maintain graft function. Any manipulation that decreases the intensity or incidence of rejection and permits the use of lesser amounts of immune suppression will result in a decreased incidence of opportunistic infection. The risk of infection and rejection are inextricably linked in a causal relationship via the net state of immune suppression. If you have viral infections your immune function is depressed. If you're taking more cyclosporine your immune function is suppressed. And that net state is going to help determine the risk of activation of retroviruses and other pathogens that may be present in donor tissues. We are able to predict quite well in the clinical situation the risk of infections following transplantation. There is a time line that we utilize to predict what infections are important. In the first month following transplantation the risk of infection is primarily those of technical complications, hematoma, indwelling catheters. The organisms of concern are those that came with the transplant or nosocomial pathogens. The surgery is unforgiving with small mistakes that are major sources of infection in transplantation. But in that first month following transplantation we're talking about familiar organisms, bacteria, fungi, parasites. In the subsequent period, the one to six month period after transplantation, we begin seeing those opportunistic pathogens, the immuno-modulatory viruses . These are not the retroviruses. These are cytomegalovirus, herpes simplex, hepatitis, and large DNA viruses that could be present very easily in a xenograft tissue. But these are the pathogens that contribute to immune suppression in the host and make them more susceptible to other opportunisticinfection. The advantage, however, is that we are able to prevent infection during the first one to six months following transplantation by using antibiotic prophylaxis.

How do we develop a strategy that encompasses some of these concepts to develop donor animals that will minimize the risk of infection in xenograft transplantation. What can we do prospectively to learn something in a research mode that makes this a safer procedure? We started looking at the miniature swine colony of David Sachs' laboratory in the Transplantation Biology Research Center at Massachusetts General Hospital in which large animal bone marrow transplant models were under development with a view towards inducing tolerance as opposed to using long term immunosuppression for xenograft transplantation. This strategy is very promising. However, given the predictable period of neutropenia, the risk of bacteremia or of other infections is enhanced initially. This makes any subsequent transplantation surgery even more unforgiving in terms of infectious risks.

The incidence of bacteremia or sepsis was easily addressed by eliminating the routine use of broad spectrum antibiotics. In this situation, we were able to avoid selection of antimicrobial-resistant flora by being careful, by being very narrow in the use of antibiotics. And we reserve broader spectrum treatment for documented infections. We quickly learned that resistance to anti-microbial agents and fungal infections emerge rapidly and they spread very quickly within a colony. Within three to five days, every single animal in a colony becomes infected with pathogens newly introduced into the colony. The microbiologic identification of porcine organisms was undertaken in collaboration with Dr. Sachs as a part of a microbiologic screening effort in the miniature swine colony. However, the use of a clinical microbiology lab for this purpose is fraught with problems. This type of laboratory, though excellent, frequently cannot speciate or isolate porcine organisms. It is therefore essential that we develop new culture techniques and new serologic techniques to identify the presence of these organisms in swine, in clinical laboratories or in human hosts. New microbiological assays are essential to the successful use of xenotransplantation.

Generally, concerns expressed about xenotransplantation have focused on retroviruses. As was noted previously, the herpes viruses are also important and need to be excluded, if possible, from potential organ donors. We know for example that each of these common viruses (e.g. CMV, hepatitis, EBV or similar agents in animals) have a direct relationship to the incidence of graft rejection and injury, and to the incidence of opportunistic infection. A good example, again from studies with Dr. Sachs' miniature swine (sponsored by BioTransplant Inc. in Boston), is porcine cytomegalovirus. While this is a benign virus in normal animals (a respiratory virus of young swine) it can be reactivated by corticosteroids, particularly in alveolar macrophages. This agent, when isolated, grows in a variety of porcine cell lines described by A.M.P. Bouillant and C.L. Kanitz a number of years ago. These individuals provided reagents which allowed the ELISA assay to be established in my laboratory at the Massachusetts General Hospital. Most (over 90%) of the miniature swine are seropositive for porcine CMV at high titers. When these animals are immunosuppressed, pulmonary alveolar macrophages secrete CMV which can be grown on a variety of permissive porcine cell lines. So we know that we can reactivate latent infection due to porcine cytomegalovirus in miniature swine. Is it important? We do know that in addition to be seropositive that CMV viral stocks have no infectivity for permissive cell lines across species lines (i.e. human for pig and porcine CMV for human).

Many of the conditions that are present in clinical transplantation will activate endogenous viruses , including retroviruses. These factors include immune suppression, graft rejection, graft-vs-host disease, the use of cytotoxic agents, and radiation exposure. The porcine retrovirus story has been clarified to some degree in our laboratory recently. In 1974, Suzuka identified a C type retrovirus from PK-15, a porcine kidney cell line. This virus is analogous to endogenous retroviruses of swine but not to primate or rodent endogenous viruses. This cloned virus was identified as a C type retrovirus. This virus causes lymphomas in wild boar but is not infective for human cell lines or mice. We have just completed the sequence of this clone in my laboratory; it appears to have some very characteristic retroviral elements. The interesting feature of this isolate is that the genes are in the wrong order compared to most of the primate viruses. Is this important? We don't know. The dissimilarity from the primate viruses may suggest, but does not prove, that this type of virus may be less infectious for humans. We are developing the tools to begin to look at these kinds of issues. These should be seen as research tools as opposed toclinical tools at the present time. This may allow some insights into the activation of retroviruses in the appropriate animal models.

One related strategy that has developed to reduce the level of rejection, particularly hyperacute rejection, following xenotransplantation, is the genetic modification of the donor animals. The effects of this type of manipulation are unclear. Some of the modifications that have been made in the complement system in transgenic swine provide those swine with receptors for human pathogens that were not previously present. So the DAF and MCP transduced swine actually have provided the receptor for coxsackievirus and for the human measles virus previously absent from these swine. That implies that if an animal caretaker has measles and goes into this colony, all of those animals may have novel susceptibility to human measles. The effect of these transgenes on viral activation or in the distribution of retroviral elements in those animals are not known. This is another important area of research for the future. The retroviral issue is very important, but we also have to be cognizant of the issues that are raised by other common viruses also. In particular, most of the activated retroviruses are nonpathogenic while cytomegalovirus and the other herpes viruses have a known importance in the field of allotransplantation and, by analogy, to xenotransplantation. It is not possible to predict the impact of retroviruses in xenotransplantation. This area has not been fully investigated in clinical allotransplant recipients. The impact on the community at large also remains unknown and unmeasurable.

There are clear advantages to xenotransplantation. The potential resistance to specific viruses may be the most important aspect of this field. The ability to put a porcine liver into a human infected with Hepatitis B or C or the ability to transfer bone marrow into an HIV infected individual with resistance to future infection has the potential to be life-saving and cost effective. As was noted previously, the absence of cellular machinery and of receptors may allow the use of xenografts in individuals who might have little sustained benefit from allotransplantation if donors could be found. The ability to avoid nosocomial infections and to time transplantation to the needs of the patient will dramatically decrease the pre- and post-transplantation costs of this procedure. The resistance of porcine organs (and baboons to hepatitis B virus) to viral infection provides another avenue of research into uses for xenograft tissues.

Porcine pancreatic transplants have been going on from swine to humans in Europe for a number of years. There have been no infectious disease problems of which we are aware. So thus far, the very limited data we have suggest that cellular transplants have not induced major infectious disease yndromes in recipients with patient 15 to 20 years after the initial transplants.

The strategy for the breeding of miniature swine for organ transplantation is simple in concept. What you cannot do is just go out and buy a pig from a local farm. It is critical for the recipient, and possibly to the community, that we be very selective in the procurement of donor animals and that we think prospectively about which organisms are potential risks to the xenograft recipient. Completely pathogen-free colonies are costly and the animals tend to grow more poorly and are more susceptible to various kinds of infections in the long run. DPF or "designated pathogen free" animals (my term) are bred to eliminate specific organisms from closed animal colonies which are maintained under barrier conditions with gown, glove and mask precautions. A closed colony may provide us with the kind of control that allows the exclusion of the major groups of pathogens which may be of concern in the immunosuppressed host. In this manner, it is possible to exclude cytomegalovirus and other pathogens that we consider to be major risks.

Which are the pathogens that are major risks? It should be possible to define the microbial flora of a closed colony to exclude those organisms not only likely to cause disease in the recipient but also those, other than endogenous retroviruses, which may pose a hazard to the population in general. First, the major zoonotic pathogens of humans can be excluded. Common organisms causing disease in transplantation recipients such as Toxoplasma gondii, Salmonella species, Sarcocystis, and Listeria monocytogenes. It is important to exclude organisms that are similar to pathogens of transplant recipients such as adenovirus or porcine cytomegalovirus. Based on experience with nosocomial spread of pathogens, the use of antibiotics will be severely restricted so as to avoid the introduction of fungi or of antibiotic resistant organisms. We will limit as much as possible viruses which lack the self editing replication machinery (reovirus, retroviruses) that are most likely to cause problems in terms of recombination. An additional list of organ-specific exclusion list might be created for specific donor organs such as Mycoplasma species in lung tissues. Given these criteria, it is possible to create a meaningful list of all potential pathogens derived from swine. For each (known) organism, an educated guess can be made based on potential importance to the transplant recipient, with particular focus on the potential for recombination or likely risks to the community at large. This process was followed in detail by myself and colleagues at BioTransplant. A list of organisms was developed that included such organisms as Listeria, Histoplasma, Aspergillus, cytomegalovirus and which can be excluded from a closed colony of miniature swine. It will be both expensive and a tremendous amount of work. Is it worth it? Well in fact its not a question of being worth it, its the only viable alternative to not having another source of organs and even so it remains an expensive proposition. But it is an achievable goal despite the cost and difficulty. I liken this process to the development of a package insert from a bottle of medicine. In other words how do you know what you're getting when you're getting a xenograft organ? The answer should be you should be getting something that is free of known or potential pathogens to the level of the present state of knowledge. We have the capability of breeding swine at least to exclude some of the risks associated with xenotransplantation. Infection will remain an important issue in transplantation as long as immune suppression is central to the maintenance of graft function.

To supplement this discussion it is worth noting that we not only want to be sure that are our animals are not infecting the animal handlers, but that the animal handlers are not transmitting organisms to these swine. As a result, criteria must be developed to screen the animal handlers to avoid or to track their transmission of organisms from humans to the individual animals. Researh strategies must be developed to investigate the epidemiology of infection in the xenograft recipient, to supplement our diagnostic tools for nonhuman pathogens, to do in-vitro and in-vivo studies in the laboratory both in terms of cross species infectivity and to focus on animal models that reproduce the xenotransplantation situation.

I started out by saying there was a direct link between immunosuppression and the risk of activation of infection. It is true regardless of what the kind of infection is and its true to a certain degree regardless of what the kind of immunosuppressive agent there are and we've got a lot of immunosuppressive agents now. The question is what can we do to increase the safety of xenograft transplantation. We are beginning to see the first vestiges of ability to do transplantation of cross species without immune suppression using tolerance protocols. While these techniques are not yet perfected, it is a major stride in the direction of reducing the level of immune suppression an the risks of all opportunistic infections. The future rests with a combination, therefore, of improved organ availability for transplantation, derivation of source animals with defined microbial flora, and the advancement of strategies which reduce the level of immune suppression needed to maintain graft function in the xenograft recipient. This is an exciting prospect, and one important enough to be the focus of a major research effort for years to come.

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Xenosis from Swine: Assessing the Infectious Risks of Xenotransplantation