[Table of Contents]

Volume: 27S1 • July 2001

Report of the Xenotransplantation Surveillance Workshop
Infection Control Database and Sample Archiving

I: INTRODUCTION

Background to the Current Regulatory Framework for Xenotransplantation in Canada

Xenografting is legally permissible in Canada only upon authorization of a clinical trial application, such as an Investigative New Drug (IND) application or an Application for Investigational Testing - Medical Devices, to the Therapeutic Products Programme* of Health Canada (HC)^(2,3). No special access program for xenografts is allowed in Canada because of the complexity of establishing risks and safety to third parties⁽³⁾. Some xenografts, such as extracorporeal perfusion devices, may more resemble medical devices than drugs, whereas others fall more under the description of a drug (biological drugs) or cellular transplantation^(2,3). Accordingly, both sets of regulations have to be taken into account when surveillance for xenotransplantation clinical trials is considered. In Canada, fixed pig valves are not considered xenografts as they are not viable⁽⁴⁾.

Adverse Event Reporting in Canada

When clinical trials on new therapeutic products are conducted to address safety and efficacy, annual reporting on patient outcomes and immediate reporting of adverse events (AEs) to HC are mandatory. For fatal and life-threatening unexpected adverse drug reactions (ADRs), the initial report must be sent to HC within 7 calendar days. All other serious and unexpected ADRs must be sent within 15 calendar days⁽⁵⁾. For serious AEs occurring during clinical trials of medical devices, HC must be informed within 72 hours⁽⁶⁾. Generically, once approval to market has been obtained, the reporting of serious drug-related AEs is voluntary, whereas for medical devices AEs must be reported within 10 days⁽⁷⁾.

In the draft Proposed Canadian Standard for Xenotransplantation (PCSX)⁽⁸⁾, it is recommended that registration of the xenograft recipient occur within 72 hours and that all serious AEs be reported immediately (although time lines are not made explicit). Although there are, as yet, no regulations referring to the proposed standard, an interim guidance document could be issued for xenotransplantation clinical trials.

According to the proposed standard, there would have to be lifelong monitoring of recipients, and this would exceed the time lines of clinical trials. Therefore, it is clear the existing regulations, at least for drugs, are not appropriate for xenotransplantation surveillance. In the U.S., the Public Health Services (PHS) Guideline on Infectious Disease Issues in Xenotransplantation⁽⁹⁾ was recently revised⁽¹⁰⁾. It now states that records and specimens on source animals and recipients should be kept for 50 years rather than for life⁽¹⁰⁾. This information and sample archive requirement were subject to public comment⁽¹¹⁾. Given the lengthy time of the recommended U.S. follow-up, further clarification on the duration of follow-up in Canada is clearly needed. This is especially true if the xenograft protocol under consideration is reviewed as a biological drug, for which (unlike medical devices) post-market reporting of adverse events is voluntary.

In Canada, precedents have already been set for enhanced surveillance schemes. For example, in February 2000, new guidelines for reporting AEs associated with vaccine biological products were released⁽¹²⁾. The Division of Immunization, Bureau of Infectious Diseases, collaborates with the Vaccines Division of the Bureau of Biologics and Radiopharmaceuticals. The Division of Immunization retains the responsibility for conducting post-marketing surveillance activities and maintains the Vaccine Associated Adverse Event Surveillance System (VAAESS). Not only was the definition of vaccine-associated adverse event (VAAE) expanded and more clearly defined in the February guideline but, as well, an Advisory Committee on Causality Assessment (ACCA) was set up in order to determine whether a particular AE was related to use of the vaccine and whether it merited further investigation⁽¹²⁾. Reporting of VAAEs from health care practitioners is voluntary (except in Ontario, where it is mandatory) and is usually channeled through provincial health authorities. However, serious VAAEs reported to manufacturers must be submitted to HC within 15 days and, for less serious AEs, within 30 days (i.e. reporting to HC is mandatory for manufacturers).

The Canadian policy on INDs (i.e. the drug regulations) states that the serious reportable AEs must be unexpected⁽⁵⁾. For xenotransplantation clinical trials, however, probably all deaths and serious changes in health should be reported immediately irrespective of cause. This would be more in line with the enhanced surveillance system in place for vaccine products⁽¹²⁾. As well, it would be reasonable to expand the definition of immediately reportable xenotransplantation-associated adverse events to include graft failure, cancer, and other disabilities related to immunosuppression, since these would be related to the xenograft protocol, though not necessarily to infectious diseases. The PCSX captures this broader definition of adverse event⁽⁸⁾.

The definition of reportable AEs for xenotransplantation is very important. Not surprisingly, it is still under heavy debate internationally, as is the definition of xenograft. In terms of the latter, the U.S. PHS recently amended its definition of xenograft to include not only live cells, tissues, or organs from a nonhuman source but also human body fluids, cells, tissues, or organs that have had ex vivo contact with them⁽¹⁰⁾. Thus, this new definition covers skin substitutes - i.e. human skin that is grown on nonhuman fibroblasts - and applies to several hundred patients treated in the U.S. Whether fixed porcine valves should be considered to be xenografts has been debated by the Council of Europe (Dr. Larry Whitehouse, personal communication), but the Council's definition⁽¹³⁾ does not currently include them. Nevertheless, it is important to determine the level of residual risk, if any, derived from the implantation of fixed porcine valves as part of the pre-clinical risk assessment process, despite the fact that no AEs have been reported so far⁽¹³⁾.

Potential Attributes of an Enhanced Surveillance Scheme

The xenotransplantation workshop itself was not set up to determine whether enhanced surveillance was necessary but, rather, what it would comprise, given the necessary resources. A national enhanced surveillance scheme for xenotransplantation might involve some of the following:

• national patient registry and adverse event reporting database;
• national specimen archiving site with a database for animal source and recipient materials and including specimens from any investigations of close contacts or health care workers;
• national testing and sample monitoring laboratory facilities (linked to the specimen archiving database) for active surveillance and for investigations/follow-up;
• minimal exclusion and inclusion criteria to reduce infectious disease risks to recipients, their offspring, and third parties; and to also enhance the likelihood of lifelong compliance with monitoring, personal infection control, and safe sex practices;
• national committee of experts to oversee xenotransplantation surveillance and testing (this may or may not be separate from the existing Expert Advisory Committee on Xenotransplantation Regulation, which deals with problems encountered during the approval process for xenotransplantation clinical trial applications);
• national emergency preparedness procedures and response team for outbreaks in collaboration with provincial, territorial, and local health authorities;
• national inspection team (before and after clinical trial approval or for specific investigations);
• careful and precise definitions of "xenograft", "serious adverse events", "other adverse events", and "suspected xenozoonosis";
• clear time lines for reporting to HC and clarification of duration of follow-up if "lifelong" is too broad, as may be detailed in an interim guidance document.

Enhanced Xenotransplantation Surveillance in Other Nations

In the U.S., the PHS has continued the development of a national xenotransplantation database that will monitor xenotransplantation patients on a lifelong basis and is exploring an option to develop a central biologic specimen archive. For now, the revised PHS Guideline recommends that sponsors should archive materials designated for use by the PHS. Source tissues will be kept in archives for at least 50 years for future reference. In addition, the U.S. Department of Health and Human Services has established a 15-member Secretary's Advisory Committee on Xenotransplantation, which will keep the public informed, serve as a public sounding board, advise the Department on the current state of knowledge regarding xenotransplantation, and review current and proposed xenotransplantation clinical trials. To date, the U.S. Food and Drugs Administration (FDA) has not announced an enhanced xenotransplantation surveillance scheme over and above what is required for bioligical drug clinical trials in general. Nevertheless, in 1997 it did place all porcine clinical trials on hold until sponsors could address certain issues, such as the testing of all recipients for porcine endogenous retroviruses (PERVs). As well, source animal facilities and the clinical animal sites do require accreditation. Further guidance from the U.S. FDA on requirements for xenotransplantation clinical trials can be found at http://www.fda.gov/cber/gdlns/clinxeno0201.pdf.

The U.K. situation is different, in that no clinical trial applications have yet been approved. The United Kingdom Xenotransplantation Interim Regulatory Authority (UKXIRA) has released a draft document for consultation called the Draft Report of the Infection Surveillance Steering Group of the UKXIRA⁽¹⁴⁾. Three clinical trial applications have been submitted to the UKXIRA, of which one was subsequently withdrawn; the other two were not accepted because of lack of supporting data.

The Issue of Endemic Viruses

The issue of testing and exclusion of pig herds bearing endemic viruses such as porcine cytomegalovirus (pCMV) requires further clarification. WHO recommendations⁽¹⁵⁾ state that herds should be free of certain viruses, including even those not established to be zoonotic, such as pCMV, as their transmission (or pathogenicity) may be enhanced under xenotransplantation protocols. While this is intended to mean that exclusion criteria should address pig infectious agents not known to be transmitted to humans but for which the risk of transmission may be facilitated through xenotransplantation⁽¹⁵⁾ (Clara Witt, personal communication), it would not necessarily preclude xenotransplantation. However, all herds apparently harbour pCMV and other endemic viruses, such as other herpesviruses, hepatitis E virus, circoviruses, parvoviruses, and papillomaviruses, which generally do not cause significant disease in their natural hosts⁽¹⁶⁾. Whether some or any of these endemic viruses can cross the species barrier under xenotransplantation conditions remains unknown. Moreover, if transgenic organs, tissues, or cells are used or if recipients are immunosuppressed, this is likely to significantly increase the risk of transmission or pathogenicity respectively.

An initial risk assessment may be possible by examining samples from occupational exposures to pig blood and medical exposures of biologic drugs or medical devices derived from pigs. However, this may not provide sufficient data to predict risk when the recipient of a xenograft is immunosuppressed or the material is derived from a transgenic animal. Such an assessment is likely to underestimate rather than overestimate risk, but would be valuable as a starting point.

A good example of variable risk dependent on the immunosuppression status of the host is hepatitis E. Although human hepatitis E virus usually involves only a temporary hepatitis, which does not lead to a chronic carrier state, an unusually high mortality rate (about 20%) occurs in third trimester pregnancy in humans, associated with vertical transmission and fetal demise⁽¹⁷⁾. Swine hepatitis E virus has 97% homology to some strains of human hepatitis E virus isolated in non-endemic countries such as the U.S. It remains to be determined whether swine hepatitis E virus, which is likely zoonotic to humans, results in an increased risk of mortality associated with pregnancy in humans. Its potential pathogenicity in immunosuppressed xenograft recipients would also need careful evaluation.

The revised U.S. PHS guideline recommends that for any infectious agent known or suspected to be in the source herd or animal, active monitoring of the recipient be done, especially in the immediate post-xenograft period⁽¹⁰⁾. Thus, while the guideline has not specifically identified which endemic porcine viruses need to be excluded from the herd, it does suggest that prospective "active" risk assessment should be done to determine whether an endemic infectious agent might need to be excluded in the future.

The Issue of Endogenous Viruses

The unknown risk of endogenous viruses, such as PERVs, has received far more attention and scrutiny than endemic pig viruses. This may be in part because HIV-1, a retrovirus, became established as a zoonotic pathogen when it jumped the species barrier, probably from chimpanzees to humans⁽¹⁸⁾. In this case it is the relatively high rate of sexual transmission that contributed most to the pandemic⁽¹⁸⁾.

In March of 1997, Patience et al. reported on the existence of PERVs that can infect human cells in vitro⁽¹⁹⁾. The following year they reported that once the endogenous retrovirus gained entry to human cells and replicated, the enveloped virions then produced were no longer susceptible to complement-mediated lysis by human serum, and infectivity rates increased with this change⁽²⁰⁾. Human serum contains naturally occurring antigalactose antibodies, which react with terminal galactose residues on viral proteins when the virus replicates in non-human cells. This suggests that a higher rate of transmission of PERVs and probably other enveloped viruses may occur if transgenic sources of animal organs, cells, or tissues are used for xenotransplantation. Denner et al., in 1998, reported that peptides in PERVs, which are at a site in the envelope protein known to be immunosuppressive in general for retroviruses, were found to be immunosuppressive in vitro⁽²¹⁾. Thus, PERV infection in humans has the potential to lead to immunosuppression in vivo. Given the difficulty in excluding PERVs from pig source animals, since the retrovirus is contained within the pig genome (i.e. is endogenously present), the question is, do pig cells, tissues, or organs release functional "infective" virions? If they do, what is the risk for cross-species transmission to humans? Finally, will PERVs exhibit significant rates of human-to-human transmission through sexual contact, the blood supply, or other means?

In many instances, endogenous retroviruses are inactive (as a result of genetic lesions), they are expressed but incapable of producing infective virions, or they are latent until activated by a number of agents, such as ionizing radiation, stress, inflammation, or potent stimulators of proliferation and/or activation (mitogens), etc. Thus, the findings by Martin et al. that infectious PERVs are released from porcine aortic endothelial cells without mitogenic or other stimulation²² and, more recently, by Van der Laan et al.⁽²³⁾, that they are released from pig islet cells when xenotransplanted into NOD/SCID (diabetic/immunosuppressed) mice suggest that the risk of retrovirus transfer to humans after xenotransplantation is at least theoretically possible. They also suggest that the screening of serum, biopsies, or preferably tissue samples of pig herds with sensitive techniques will detect PERV virions, making it difficult to obtain pig source animals that do not excrete PERV virions. Indeed, others have found that there is variability in the production of PERVs in blood cells from different pig strains⁽²⁴⁾, and that in Specific Pathogen Free (SPF) herds there is variability of viral mRNA load with tissue sources (kidney being the highest producer, then liver, lung, and heart)⁽²⁵⁾. Most notably, the SPF herds expressed PERV mRNA at equivalent or higher levels than conventional herds. This indicates, as expected, that the use of SPF herds does not reduce the risk of PERV transmission to humans.

The finding of the presence of an infectious agent in all or most potential source animals is necessary but not sufficient to indicate the level of risk of a) transmission to humans, b) disease causation, or c) the potential for an epidemic. Thus, while there is an apparent risk of transmission of PERVs to humans with xenotransplantation protocols, the question is, how often does it happen? A number of investigators have tried to address this issue^(26-31). So far there is no unequivocal evidence that PERVs have infected humans or replicated in human hosts in vivo^(26-31). However, in splenic perfusion models, 23% of the recipients followed up had circulating pig cells (referred to as a microchimerism) containing latent PERVs⁽²⁶⁾. The long-term consequences of microchimerism are unclear, although some individuals had had their xenograft exposure 8 years before⁽²⁶⁾. In another study, involving 10 patients with implanted fetal pig islet cells, five had microchimerism at 6 months or longer⁽³¹⁾. In neither study were antibodies to PERVs detected, suggesting that no release of infectious particles took place; however, it is possible, though not usual, to have an infection without antibody production, such as early in the course of infection during the window period, or as a result of tolerance induction. Nevertheless, there is no clear evidence for PERV replication or infection in human hosts even when microchimerism might exist^(26,31), although there are caveats to this interpretation, since transgenic xenograft sources were not evaluated. The report of active PERV infection in a murine model⁽²³⁾ gives great concern about a transpecies infection in humans, however.

If prospective testing of appropriate tissue samples, such as kidney or epithelial cells, were carried out instead of retrospective testing of serum or blood samples, positive results might be found under certain conditions, such as in immunosuppressed human xenograft recipients or in those receiving transgenic xenografts. Recently, it was reported that 68% (17 of 25) NOD/SCID mice xenotransplanted with pig islet cells had microchimerism, and all 8 mice that could be evaluated had evidence of PERV infection, albeit only in the tissues where microchimerism could be demonstrated⁽²³⁾. The authors suggested that pig islet xenotransplantation to humans may result in long-term exposure to replication-competent endogenous retrovirus. However, since these investigators did not exclude the possibility of pseudotyping of PERV with mouse endogenous retrovirus (which may have increased the risk of transmission), it is currently unclear whether this increased risk of transmission in mice can be extrapolated to humans.

Published Adverse Events in Xenograft Recipients

The only published autopsy report involving the death of a xenograft recipient was at 7.5 months after implantation of porcine fetal neurons into a patient with Parkinson's disease in the U.S.⁽³²⁾. The cause of death was a pulmonary embolism deemed to be unrelated to the xenotransplantation protocol⁽³²⁾. However, this patient was immuno-suppressed with cyclosporin, and there was an associated higher risk of infection. Additionally, infectious agents can be associated with pulmonary emboli. There was no indication that a follow-up investigation was done for porcine infectious agents, so it is unclear whether endogenous or endemic porcine infectious agents contributed to the patient's demise. A complete analysis of autopsy samples for endogenous and endemic infectious agents of pigs (along with source animal testing) would have been critical to address the general issue of transmission of these known viral agents to humans. An argument could be made that deaths in xenotransplantation clinical trials should be reported immediately and autopsy tissue samples fully investigated by health authorities, irrespective of perceived cause of death.

In a study of 10 Swedish patients who received transplanted fetal pig islet cells with immunosuppression, the follow-up was 4.5 years or more⁽³¹⁾. During this time two patients died of myocardial infarction, and one patient lost a renal allograft (at 2.5, 5 and 6 years respectively). No lymphoproliferative or neurologic disease was reported. It is unknown whether any of these serious adverse events were associated with xenozoonosis. Although no evidence was found from blood testing to suggest PERV infection⁽³¹⁾, the report did not indicate whether autopsy or biopsy samples were analyzed, particularly with respect to endemic and endogenous porcine viruses. Interestingly, in the same study the majority of pigs tested (9 of 12, 75%) exhibited PERV RNA in the serum correlated with demonstrable reverse transcriptase (RT) activity⁽³¹⁾. Since the patients did not show PERV RNA or RT activity in serum samples but most pigs did, this offers a measure of confidence that the patients, half of whom exhibited microchimerism, did not produce replication-competent PERVs. On the other hand, this finding in pigs suggests that the infectious disease risks related to PERVs are likely to be there irrespective of the types of organs, tissues, or cells used as xenografts. This may be a problem if some individuals already express endogenous or contract exogenous retroviruses that might contribute to pseudotyping of PERV. This is of concern as it may subsequently allow for generation of replication-competent PERVs. The possibility of endogenous retrovirus likely contaminating most or all xenografts is cause to take extra precautionary measures. If these endogenous viruses could be genetically or functionally deleted from the pig genome (of which many scientists are not hopeful), the risk of infectious disease associated with xenotransplantation is likely to be greatly reduced.

Infectious Disease Risks of Endemic and Endogenous Porcine Viruses

In summary, the risks of infection to human populations by pig endogenous and endemic viruses remain largely unknown, although there is suggestive evidence that swine hepatitis E may be transmissible to humans. Active monitoring of common pig infectious agents after xenotransplantation will be necessary to provide a risk assessment for endogenous and endemic viruses. In particular, the immediate post-xenotransplantation period (i.e. before antibodies clear the virus from patient blood), which can be anywhere from 2 to 8 weeks, would be the optimal time to investigate for viral replication by, for example, polymerase chain reaction (PCR). Close examination of the viruses that are replicating in the early post-transplantation period would at least answer the question of what the infectivity risk is of the various pig infectious agents for humans. This would indicate which viruses need to be closely monitored over the longer term in order to control and contain a potential epidemic.

As is well known, absence of data does not substantiate absence of risk. The finding of cross-species transfer of PERVs⁽²³⁾ to immunosuppressed mice in association with microchimerism raises the question of whether immunosuppressed humans receiving transgenic organs, tissues, or cells or, alternatively, recipients with microchimerism will also produce replication-competent PERVs. Furthermore, as will be discussed later, any adverse event could be linked to infection with a zoonotic agent. Without active screening, it will be difficult to determine whether the symptom relates to a potential xenozoonosis or not. Initial trials should be closely and carefully monitored⁽¹⁾. The revised draft U.S. PHS guideline also now calls for active monitoring after xenotransplantation of any pig infectious agents known or suspected to be in the source herd, including endogenous and endemic pig viruses.

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