Safety of Xenotransplantation: Development of screening methods and testing for porcine viruses

Xenotransplantation using pig cells, tissues or organs might be a promising solution to overcome the shortage for organs suitable for allotransplantation. Because of several reasons, the pig is currently the favoured donor species. However, the use of porcine xenotransplants is associated with the risk of transmitting porcine viruses to the human xenotransplant recipient. Among them porcine endogenous retroviruses (PERVs), porcine cytomegalovirus (PCMV), porcine lymphotropic herpesviruses (PLHVs), porcine circovirus 2 (PCV2) and hepatitis E virus (HEV) play a role. Some of them cause immunosuppression and a zoonotic potential of others has been supposed. Therefore the possibility of direct transmission of those viruses between pigs and humans might be possible. Strategies to avoid the transmission of those pathogens are currently of main importance to increase lifetime of the transplant and therefore to save many lives of people standing on the transplant waiting list. To select virus-free animals as putative donor pigs and to recognise transmission of pathogens to transplant recipients, sensitive detection methods are needed. In this study the prevalence and expression of these selected viruses should be investigated and assessed in order to obtain safe and healthy donor pigs for xenotransplantation studies. Therefore highly sensitive PCR-based methods, real-time PCR and real-time RT-PCR specific for all the viruses listed above, as well as immunological methods measuring virus-specific antibodies by Western blot analysis or ELISA were developed. Recombinant viral proteins were cloned, expressed and chromatographically purified as well as purified virus particles were expanded to be used as antigens. The methods were developed and optimized to screen (i) Göttingen minipigs, a well characterized pig breed which is kept in a specific-pathogen free facility, (ii) Aachen minipigs, a pig breed existing since 2013, (iii) slaughterhouse pigs from a butchery in the north of Berlin and (iv) multiply genetically modified pigs produced especially for xenotransplantation. Human-tropic PERV-A and PERV-B were found in all pigs and pig-tropic PERV-C and recombinant PERV-A/C were found in many pigs. HEV, PCMV, PLHVs and PCV2 were found in a few animals. No transmission of the porcine viruses listed above was observed during the transplantation of genetically modified islet cells into four marmosets. However, when transgenic pig hearts were transplanted into baboons, then PCMV and HEV were found transmitted, despite the fact that the donor pigs were negative when testing blood and antibody response. To avoid future transmissions of porcine viruses, more sensitive detection methods, different time points of testing, and different source materials, including oral and anal swabs, should be used. In the study sensitive and reliable methods for the detection of porcine viruses were developed and those viruses were detected in all tested pig herds. Furthermore, potentially zoonotic viruses like HEV and viruses causing immunosuppression like PCMV, PLHVs and PCV2 are present in pigs for slaughter. Although the expression of these viruses were low, the meat-producing and -processing industry should be aware of the improvement of hygienic standards. The newly developed detection methods are a prerequisite for the selection of virus-free pigs for transplantation trials as well as elimination programs based on treatment, vaccination, Caesarean delivery, early weaning and embryo transfer

When should precaution prevail? : interests in (public) health, the risk of harm and xenotransplantation.

Xenotransplantation is an example of a developing biotechnology which highlights three differing interests in the health of the public; a specific interest in enhancing the health of individuals who require a particular procedure or treatment, a wider interest in protecting the health of us all by avoiding introducing biotechnologies which risk the health of the public, and a public interest in advancing medical knowledge and treatment. Here we explore how matters of private benefit and public risk can be appropriately reconciled and consider whether ideas of public health should take a more central role when deciding whether clinical xenotransplantation should proceed. The risks of xenotransplantation are not certain but the nature of the harm to individual and public health could be severe. The concept of risk is central to our analysis as xenotransplantation threatens potential future harm as well as possible benefit. We argue that it is sometimes legally and ethically necessary for the state to act in advance to protect the health of the public, and that xenotransplantation is such a case. In reaching this conclusion we adopt a precautionary approach; an approach which we modify by Mill’s harm principle

An approach to the control of disease transmission in pig-to-human xenotransplantation.

Abstract: Although several major immunologic hurdles need to be overcome, the pig is currently considered the most likely source animal of cells, tissues and organs for transplantation into humans. Concerns have been raised with regard to the potential for the transfer of infectious agents with the transplanted organ to the human recipient. This risk is perceived to be increased as it is likely that the patient will be iatrogenically immunocompromised and the organ-source pig may be genetically engineered in such a way to render its organs particularly susceptible to infection with human viruses. Furthermore, the risk may not be restricted to the recipient, but may have consequences for the health of others in the community. The identification of porcine endogenous retroviruses and of hitherto unknown viruses have given rise to the most concern. We document here the agents we believe should be excluded from the organ-source pigs. We discuss the likelihood of achieving this aim and outline the potential means by which it may best be achieved

How, where and when to screen for porcine cytomegalovirus (PCMV) in donor pigs for xenotransplantation

Porcine cytomegalovirus (PCMV), that is actually a porcine roseolovirus (PRV), is a common herpesvirus in domestic pigs and wild boars. In xenotransplantation, PCMV/PRV has been shown to significantly reduce the survival time of pig kidneys and hearts in preclinical trials with different non-human primates. Furthermore, PCMV/PRV has been transmitted in the first pig to human heart xenotransplantation and contributed to the death of the patient. Although transmitted to the recipient, there is no evidence that PCMV/PRV can infect primate cells including human cells. PCMV/PRV is closely related to the human herpesviruses 6 and 7, and only distantly related to the human CMV (HCMV). Antiviral drugs used for the treatment of HCMV are less effective against PCMV/PRV. However, there are well described strategies to eliminate the virus from pig facilities. In order to detect the virus and to eliminate it, highly sensitive detection methods and the knowledge of how, where and when to screen the donor pigs is required. Here, a comparative testing of organs from pigs of different ages using polymerase chain reaction (PCR)-based and immunological methods was performed. Testing young piglets, PCMV/PRV was detected effectively by PCR in blood, bronchoalveolar lavage fluid, tonsils and heart. In adult animals, detection by PCR was not successful in most cases, because the virus load was below the detection limit or the virus was in its latent stage. Therefore, detection of antibodies against selected recombinant proteins corresponding to epitopes detected by nearly all infected animals in a Western blot assay is advantageous. By contrast, immunological testing is not beneficial in young animals as piglets might have PCMV/PRV-specific antibodies obtained from their infected mother via the colostrum. Using a thoughtful combination of PCR-based and immunological methods, detection of PCMV/PRV in donor pigs for xenotransplantation is feasible and a controlled elimination of the virus by early weaning or other methods is possible

Full Issue: Volume 16, Number 1, Fall 2022

Complete .pdf file of Volume 16, Number 1 of The Science Journal of the Lander College of Arts and Sciences. Published Fall 2022

Distribution Of Porcine Endogenous Retrovirus (PERV) Variants In Domestic And Feral Pig

Xenotransplantation is considered an alternative to allotransplantation to relieve the current shortage of human organs. Due to their similar size and physiology, the organs of pigs are of particular interest for this purpose. Endogenous retroviruses are a result of integration of retroviral genomes into the genome of infected germ cells as DNA copies (proviruses), which are then carried in all cells of the offspring of the organism. Porcine Endogenous Retroviruses (PERVs) are of special concern because they are found in pig organs and tissue that might be used for xenotransplantation. PERV proviruses, already incorporated into the pig’s genome, can be induced to replicate and recombine in pigs, and have been shown to infect human cells in vitro. There are three classes of PERVs, namely PERV-A, PERV-B, and PERV-C. PERV-A and PERV-B can infect human cells in vitro and can recombine with PERV-C, resulting in a recombinant virus with a higher rate of replication in pig and human cell lines. In this study, a PCR based analysis of 50 domestic and 35 feral pigs was carried out to study the distribution of PERVs A, B, and C. PERV-A and PERV-B were universal in both domestic and feral pigs. The feral varieties of pigs displayed a higher frequency of 85.67% of PERV-C compared to 42.00% in domestic pigs. However, comparative study of presence of PERVs A, B, and C in different breeds of domestic pigs shows there is variation in distribution among breeds, and among individuals of same breeds. From the results of this study, I hypothesize that presence of endogenized PERV genomes in individuals of the same breed is dependent on genetic properties of individual pigs

Pig tales, human chimeras and man-made public health hazards : an ethical analysis of xenotransplant benefits and risks

Xenotransplantatie wordt voorgesteld als een mogelijke oplossing voor het orgaantekort, alsook voor andere aandoeningen waarvoor momenteel geen of weinig behandelingen bestaan, zoals bepaalde neurologische ziektes en diabetes Type 1.De opzet van dit doctoraat was bij te dragen aan het ruime debat over hoe men de potentiële voordelen van deze technologie kan laten opwegen tegenover de unieke risico’s en kosten die met de technologie gepaard gaan