Risk In Xenotransplantation Research Articles

Communicating About Xenotransplantation: Models and Scenarios

Xenotransplantation entails using organs from genetically modified animals as a way to solve the shortage of human organs for transplantation. As with other novel technologies, if xenotransplantation is to be judged fairly, proponents must explain its complex, uncertain, and unfamiliar risks and benefits. Xenotransplantation's risks include the possibility of a recombinant virus infecting human transplant recipients, potentially causing an epidemic of an unfamiliar disease. Using materials vetted by scientific experts, we communicated the variables and relationships determining this risk in three formally equivalent formats: (a) a graphic model, (b) scenarios structured by the graphic model, and (c) both the model and the scenarios. Participants were randomly assigned to receiving one set of materials. They rated them as equally clear and studied them equally long, suggesting similar ease of cognitive processing. Compared to participants receiving the scenarios, those who received the graphic model better identified causes and effects of the risk, and saw less risk of xenotransplantation. Participants who received both the model and the scenarios generally showed intermediate responses. The study demonstrates a general procedure for developing and evaluating formally equivalent graphic and scenario communications regarding highly uncertain risks. In this application to xenotransplantation, presenting a graphic representation improved people's understanding of the risk.

The regulation of xenotransplantation in Germany and in the European Union

Since the middle of the 1990, xenotransplantation has been the object of regulatory work. In this context, the point of main emphasis is the issue of protection of the individual and the general public against the risks of xenotransplantation, especially against the risks of infection associated with xenotransplantation. International organizations, such as the WHO and the Council of Europe have issued a series of publications dealing with xenotransplantation (inter alia, [1-3] below), which, however, are not legally binding. Yet, there are legally binding provisions at EU level. First the general regulations on medicinal products are applied [4-6]. Provisions specifically relating to xenogeneic medicinal products are included in Part IV Annex I of Directive 2001/83/EC [4] and in Directive 2001/20/EC [5]. In addition, the document entitled “Points to Consider on Xenogeneic Cell Therapy Medicinal Products” [7] by the EMEA comprises basic considerations on the development and evaluation of xenogeneic cell therapy medicinal products. Regulation (EC) No 1394/2007 on advanced therapy medicinal products [8] which entered into force at the end of 2007, will soon introduce further specific provisions concerning the authorisation, supervision, and pharmacovigilance of xenogeneic medicinal products as well. For the purpose of adjustment to the gain in scientific knowledge and harmonization of the regulatory framework, currently various Community rules are being further developed and/or elaborated. Moreover, a Committee on Advanced Therapies is to be established at the EMEA. In Germany, the legal framework of xenotransplantation is substantially determined by Community rules, particularly by ordinances and directives. Ordinances directly apply to all member states and thus they have the force of law. However, directives first have to be incorporated into national law, which, as far as the above-mentioned directives are concerned, was substantially effected in the German Medicinal Products Act [9] and in the German GCP-Ordinance [10]. Moreover, in the context of xenotransplantation further provisions are relevant, inter alia, provisions of the genetic engineering legislation, of the animal welfare legislation, the professional standards and the code of professional conduct of physicians and of the protection against infection legislation.

Genomic presence of recombinant porcine endogenous retrovirus in transmitting miniature swine

The replication of porcine endogenous retrovirus (PERV) in human cell lines suggests a potential infectious risk in xenotransplantation. PERV isolated from human cells following cocultivation with porcine peripheral blood mononuclear cells is a recombinant of PERV-A and PERV-C. We describe two different recombinant PERV-AC sequences in the cellular DNA of some transmitting miniature swine. This is the first evidence of PERV-AC recombinant virus in porcine genomic DNA that may have resulted from autoinfection following exogenous viral recombination. Infectious risk in xenotransplantation will be defined by the activity of PERV loci in vivo.

Infectious risk in xenotransplantation

Purpose of review Xenotransplantation of tissues from swine into humans poses the threat of transfer of known and unknown porcine microorganisms to xenograft recipients and to the general population. Recent data are reviewed that offer a perspective on the risk for, and prevention of, xenozoonotic infections. Recent findings Progress has been reported on the identification, monitoring, and exclusion of exogenous porcine viruses from the donor pig. Human-tropic porcine endogenous retrovirus appears to be a recombinant of porcine endogenous retrovirus types A and C. Published observations of porcine endogenous retrovirus infection of human tissues in murine models appear to be artifacts of pseudotyping of porcine viruses by murine retroviruses and may overestimate the risks posed by xenotransplantation. The role of porcine endogenous retrovirus-encoded xenoantigens in xenograft rejection is under investigation. Summary A combination of barrier derivation, early weaning, and genetic engineering may be needed to exclude potential pathogens from xenograft tissues. Mouse models are limited in utility for retroviral studies of infectivity for human tissues by the presence of pseudotyping murine leukemia viruses. Protocols for microbiologic monitoring of patients and their social and sexual contacts must precede clinical trials. Techniques exist to monitor potential infectious complications after xenotransplantation so as to allow well monitored clinical trials.

Phylogeny, recombination and expression of porcine endogenous retrovirus γ2 nucleotide sequences

Endogenous retroviral sequences in the pig genome represent a potential infectious risk in xenotransplantation. Porcine endogenous retrovirus (PERV) gamma sequences described to date have been classified into several families. The known infectious, human-tropic PERVs have been assigned to the PERV gamma1 subfamilies A, B and C. High copy numbers and full-length clones have also been observed for an additional family, designated PERV gamma2. The aim of this study was to examine the PERV gamma2 family by analysis of retroviral pro/pol gene sequences. The proviral load was observed to be similar among various pig breeds. Although clones harbouring an open reading frame in the examined region were found, analysis of published large PERV gamma2 clones revealed multiple deleterious mutations in each of the retroviral genes. Various recombination events between gamma2 genomes were revealed. In contrast to PERV gamma1, phylogenetic analyses did not distinguish defined subfamilies, but indicated the independent evolution of the proviruses after a single event of retroviral amplification. Expression analysis showed large PERV gamma2 transcripts and variable transcription in several tissues. Analysis of the two published gamma2 env gene sequences observed the partial lack of the receptor-binding domain. Overall, this study indicated the low infectious potential for PERV gamma2.

A NOVEL STRATEGY TO PREVENT PERV TRANSMISSION TO HUMAN CELLS BY REMODELING THE VIRAL ENVELOPE GLYCOPROTEIN

P1065 Aims: Xenotransplantation offers a potential solution to the shortage of available organs for transplantation. The swine represents an ideal source of such organs. However, the discoveries that pig endogenous retroviruses (PERV) can infect human cells in vitro and SCID mouse tissue in vivo have stimulated considerable discussion concerning infectious risk in xenotransplantation. In addition, in pigs, at least 50 proviral copies of PERVs are present in the genome. Therefore, pigs that are completely devoid of all PERV related elements will never be a possibility.vIn our previous work, we demonstrated that the high mannose type of N-glycan of the envelope glycoprotein is closely related to PERV infectivity with respect to human cells. In this study, we addressed the effects of the remodeling pig cell-surface glycoproteins, especially high mannose type N-glycan, on the susceptibility of PERV by alpha-1,2 mannosidase IB (Man-IB), which converts Man9GlcNAc to Man5GlcNAc, and alpha- 3-D-mannoside-beta-1,2-N-acetylglucosaminyltransferase I (GnT I) which catalyzes the first step in the conversion of oligomannose to complex or hybrid N-glycans of glycoproteins. Methods: Pig endothelial cells (PEC) were transduced with the LacZ gene with the packaging signal of murine leukemia virus (MuLV) under the control of the long terminal repeat of MuLV by a pseudotype infection to produce PEC(Z). The PEC(Z)s were then further infected with PERV subtype B (PERV-B) to produce PEC(Z)/PB.The PEC(Z)/PBs were next trasnfected to the Man IB or GnT-I gene by the lipofection method in order to reduce the levels of high mannose type of N-glycans. Culture supernatants of infected cells were inoculated to HEK293 cells. The inoculated cells were histochemically stained and the lacZ-positive blue foci (Blue forcus Forming Unit: BFU) were counted. Results: PERV transmission from the PEC(Z)/PB with Man IB to HEK 293 cells was reduced in comparison with control PEC(Z)/PB in each of the three independent cell lines (277.9±42.5 vs. 91.6±15.0, 373.0±65.7 vs. 101.9±65.7, 215.1±25.7 vs.63.2±8.6 BFU/well; p<0.01). The reduction rate of infectivity was approximately 30% (32.6±2.8, 30.1±2.5, 30.1±2.5%). On the other hand, GnT-I transfection to the PEC(Z)/PB downregulated the PERV transmission in three independent cell lines (766.0±80.7 vs. 191.4±32.2, 919.1±69.9 vs. 404.2±54.4, 386.5±95.9 vs.69.5±7.7 BFU/well; p<0.01). The reduction rate of infectivity was also approximately 30% (24.8±3.2, 40.9±5.1, 29.4±4.5%). The co-transfection of Man IB and GnT-I experiments are under investigation. Conclusions: The transfection of the Man IB and GnT-I genes to pig cells is quite effective in reducing PERV infectivity to human cells. The results suggest that our strategy could be useful as a new approach for solving PERV infections in clinical xenotransplantation.

Bioethical and legal perspectives on xenotransplantation

As scientific research continues to push forward the once seemingly insurmountable barriers of medical research, xenotransplantation has been viewed as a means to overcome the current and predicted future shortages of human donor organs. The current review of Australia's xenotransplantation guidelines by the National Health and Medical Research Council provides for a timely evaluation of the scientific merits, ethical dilemmas and legal implications of this technology. This paper contends that even if the scientific barriers of xenotransplantation were successfully circumvented, a myriad of ethical and legal questions remain. These questions include, but are not limited to, the use of 'donor' animals as a source of surrogate human tissues, the ability of seriously ill patients to provide fully informed consent, and the requirement of life-long monitoring of patients and third parties. This paper contends that the ethical questions and potential legal implications of xenotransplantation are so great as to outweigh any potential benefits that may be derived from this technology. Further, it is argued that as the public health risks of xenotransplantation have the potential to transcend sovereign borders and may remain latent for extended periods of time, a global regulatory approach is required in order to protect the global community and fundamental human rights.

Reduced sensitivity to human serum inactivation of enveloped viruses produced by pig cells transgenic for human CD55 or deficient for the galactosyl-alpha(1-3) galactosyl epitope.

Complement activation mediated by the major xenogeneic epitope in the pig, galactosyl-alpha(1-3) galactosyl sugar structure (alpha-Gal), and human natural antibodies could cause hyperacute rejection (HAR) in pig-to-human xenotransplantation. The same reaction on viruses bearing alpha-Gal may serve as a barrier to zoonotic infection. Expressing human complement regulatory proteins or knocking out alpha-Gal epitopes in pig in order to overcome HAR may therefore pose an increased risk in xenotransplantation with regard to zoonosis. We investigated whether amphotropic murine leukemia virus, porcine endogenous retrovirus, and vesicular stomatitis virus (VSV) budding from primary transgenic pig aortic endothelial (TgPAE) cells expressing human CD55 (hCD55 or hDAF) was protected from human-complement-mediated inactivation. VSV propagated through the ST-IOWA pig cell line, in which alpha-galactosyl-transferase genes were disrupted (Gal null), was also tested for sensitivity to human complement. The TgPAE cells were positive for hCD55, and all pig cells except the Gal-null ST-IOWA expressed alpha-Gal epitopes. Through antibody binding, we were able to demonstrate the incorporation of hCD55 onto VSV particles. Viruses harvested from TgPAE cells were relatively resistant to complement-mediated inactivation by the three sources of human sera tested. Additionally, VSV from Gal-null pig cells was resistant to human complement inactivation. Such protection of enveloped viruses may increase the risk of zoonosis from pigs genetically modified for pig-to-human xenotransplantation.

Analyses of prevalence and polymorphisms of six replication-competent and chromosomally assigned porcine endogenous retroviruses in individual pigs and pig subspecies

As porcine endogenous retroviruses (PERV) productively infect human cells in vitro, they pose a serious risk in xenotransplantation and xenogeneic cell therapies. We have analyzed the prevalence of six well-characterized full-length PERV, five of them being replication-competent and four of them being chromosomally assigned (J. Virol. 75 (2001) 5465; J. Virol. 76 (2002) 2714). These analyses revealed a heterogeneous distribution of PERV among individuals and, as no PERV is present in every pig, it seems feasible to generate pigs free of functional PERV by conventional breeding. Conversely, as PERV are polymorphic, single proviruses may have escaped detection and this kind of assay must be performed for every herd used in xenotransplantation or xenogeneic cell therapies. In addition, specific proviruses show internal point mutations which significantly affect their replicational capacities. As there are two different types of PERV LTR structures showing varying levels of transcriptional capacity (J. Virol. 75 (2001) 6933), an analysis of 21 distinct chromosomal locations revealed that PERV which harbor highly active LTRs with repeat elements in U3 are dominant.

Characterization of porcine endogenous retrovirus gamma pro-pol nucleotide sequences.

Endogenous retroviral sequences in the pig genome (PERV) represent a potential infectious risk in xenotransplantation. All known infectious PERV have been asssigned to the PERV gamma1 family, consisting of the subfamilies A, B, and C. The aim of the study was the concise examination of PERV gamma by the analysis of the retroviral pro-pol sequences. The analysis of 52 pro-pol clones amplified in this study revealed eight PERV gamma families. In addition to four already-described families (gamma1, gamma4, gamma5, gamma6), four novel families (gamma7, gamma8, gamma9, gamma10) were identified. Quantitative analysis of the novel PERV gamma sequences in selected breeds revealed variations in the endogenous retroviral load. Open reading frames (ORF) in the amplified proviral fragment were only found for PERV gamma1. In addition, novel ORF-containing PERV gamma1 clones consisting of hybrid sequences were revealed. Sequence comparison from published full-length PERV gamma1 clones of the PERV subfamilies A, B, and C resulted in a lack of strict correlation of the classification of pro-pol and env. The results indicated the occurrence of causative recombination events between retroviral genomes. Thus, our study on PERV gamma provides new data for the evaluation and selection of pigs intended to be used in xenotransplantation.

Generation and testing of a highly specific anti‐serum directed against porcine endogenous retrovirus nucleocapsid

Advances in xenotransplantation offer chances to alleviate the shortage of human donor organs. The discovery that pig endogenous retroviruses (PERV) can infect human cells in vitro has stimulated the discussion on infectious risk in xenotransplantation. A molecular and immunologic monitoring of xenograft recipients and of donor animals for putative infection with PERV and other microorganisms is inevitable. In this report, we describe the generation and testing of a highly specific anti-serum directed against the PERV nucleocapsid protein. The Gag amino acid (aa) sequence of PERV class B was used to define immunogenic domains by computer analysis. A peptide corresponding to the C-terminal 19 aa of the 10 kDa (p10) nucleocapsid (NC) portion of the Gag polyprotein was used to immunize rabbits. The generated serum was tested using recombinant PERV Gag protein expressed in insect cells, purified PERV virus particles and human 293 cells transfected or infected with PERV, respectively. Test methods included Western blotting, indirect immunofluorescence, immunoperoxidase assay and ELISA. The PERV anti-serum provides a tool that is instrumental for detection of a potential agent of zoonosis. It can be used for screening of donor animals and xenograft recipients in the course of xenotransplantation procedures.

Replication-competent chimeric lenti-oncovirus with expanded host cell tropism.

Baboon bone marrow was grafted into human immunodeficiency virus type 1-infected patients in the course of recent trials for AIDS treatment. Since the baboon genome harbors multiple copies of an endogenous oncovirus, chimeric lenti-oncoviruses could emerge in the xenotransplant recipient. To analyze the potential replication competence of hybrid viruses between different genera of retroviruses, we replaced most of the env gene of simian immunodeficiency virus with the env gene of an amphotropic murine leukemia virus. The hybrid virus could be propagated in human T-cell lines, in peripheral blood mononuclear cells of rhesus macaques, and in CD4- B-cell lines. Because of the expanded cell tropism, the hybrid virus might have a selective advantage in comparison to parental viruses. Therefore, emerging chimeric viruses may be considered a serious risk of xenotransplantation. A note of caution is also suggested for the use of pseudotyped lentiviral vectors for human gene therapy.

Erratum: FDA Puts the Brakes on Xenotransplants

In the news article "FDA puts the brakes on xenotransplants" by Rachel Nowak (5 May, p. 630) the description of the response of Columbia Presbyterian Medical Center in New York City to one of its investigators' proposal to test baboon-to-human heart transplants was incorrect. In fact, Columbia convened one panel of outside experts to advise its internal committees on the risks of xenotransplantation, and has since retained two outside experts as consultants to Robert Michler, the principle investigator for the xenotransplant trials. The institution also recommended that a group of experts on emerging infections be convened to examine the risk of viral transmission associated with xenotransplantation (Letters, 21 Apr., p. 349).