Safety
Ensuring a high safety profile for xenotransplantation is of primary importance in view of its possible clinical application. Indeed, there is the need to minimise the risk of potential transmission of infectious agents through this approach. Accordingly, research has been undertaken by many groups in this area. Currently, using specific pathogen-free colonies and specialised animal husbandry, it is possible to exclude the vast majority of known bacterial, viral and parasitic pathogens from pig herds. Furthermore, research conducted during the past ten years and retrospective studies in humans exposed to live porcine cells and tissues have not shown transmission through these procedures of any potential infectious agents to man, including viruses.
Nevertheless, studies assessing the safety of xenotransplantation are ongoing and are primarily focused on viruses, especially porcine endogenous retrovirus (PERV), which are encoded in the germ line DNA. In this respect, extensive investigations have demonstrated that multiple copies of PERV are integrated in the genome of all pig strains and that viral particles are produced by normal pig cells (reviewed by Fishman and Patience). Three classes of infectious PERV have been identified (PERV-A, -B and -C) and classified based on differences in receptor recognition which are responsible for differences in host range. PERVs have been isolated from both porcine cell lines and primary cells, although viral particles derived from primary pig cells are generally present in low titres and show limited replication ability, a favourable aspect with respect to the xenotransplantation of pig organs. Whilst PERV-A and PERV-B can infect human cells in vitro, the PERV-C subgroup lacks this capacity. However, it has recently been observed that in vivo recombination between PERV-A and PERV-C is possible and can produce a human-tropic recombinant virus. Nevertheless, recombinant PERV-A/C proviruses have not been identified in the germ line DNA of pigs capable of transmitting PERV. Most importantly, no evidence of human infection with any PERV, even with the recombinant PERV-A/C virus, has been reported.
As far as exogenous viruses are concerned, particular interest has been paid to the four families of herpes viruses identified in pigs: porcine cytomegalovirus (PCMV) and porcine lymphotropic virus -1, -2 and -3 (PLHV-1, -2 and -3). Although PCMV activation has been documented in pig-to-primate xenografts, causing clinical disease in the xenotransplanted organ and the detection of viral DNA in primate tissues, it does not appear to cause invasive disease in transplanted primates. Moreover, it has been demonstrated that PCMV can be effectively excluded from source pigs by early weaning. Of the three PLHV viruses identified, only PLHV-1 is associated with a lymphoproliferative syndrome similar to post-transplantation lymphoproliferative disease following allogeneic bone marrow transplantation in swine, but such a disorder has not been observed in pig-to-primate xenotransplantation.
It has been pointed out that some of the strategies aimed at minimising xenograft rejection may increase the risk of zoonoses. First, the use of immunosuppressive regimens and tolerance induction protocols may exacerbate the risk of infection from otherwise non-infectious or latent animal pathogens. Second, viruses released by cells that do not express α-Gal, such as those of α-GalT knock-out pigs, lack α-Gal epitopes on their envelope and cannot be recognised by anti-α-Gal antibodies, therefore becoming less sensitive to complement-mediated inactivation. Third, the presence of human complement regulators on transgenic pig cells may reduce complement-mediated defence mechanisms against infections. Finally, some human complement regulatory proteins are receptors for human viruses. Therefore, in genetically engineered pig lines expressing such human complement regulatory proteins, pig viruses may adapt to infect humans once porcine organs have been transplanted. However, it is reassuring that, despite all these considerations, to date, no experimental or clinical findings have supported these concerns.
In addition, it is noteworthy that PERVs and other viruses are susceptible to some of the currently available antiviral agents. Furthermore, pig lines have recently been identified which are incapable of transmitting PERV to human cells in vitro. Finally, genetic manipulation of the porcine genome may provide an additional strategy to remove the viral risk. Indeed, both specific knock outs of endogenous retroviruses or short interfering RNAs specific for PERV sequences have been proposed.
Moreover, the development of microarray-based technology capable of rapidly identifying known and as yet unidentified potential infectious agents may allow their timely identification and control in the xenotransplantation settings.
In conclusion, the described genetic procedures, in combination with controlled breeding conditions and lifelong source animal monitoring will ultimately result in the availability of source animals with a high safety profile and minimised risk of zoonoses.
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