sou · GNB3120d2 · 2000-01-01 00:00:00 — Endogenous retroviruses (PoERVS or PERVS) are spontaneously produced from certain primary and secondary porcine cell cultures or may be induced to do so by various mutagenic events (et al , et al , Todaro et al , Lieber et al , Moennig et al , Strandström et al , Bou illant et al , Lieber et al ). Initial studies failed to show that porcine endogenous retroviruses were able to infect human cells. In 1996 one of us () reported to the FDA the infection of human and 293 cells by PK15 cells and independently and colleagues demonstrated the infection of human cells by PoERVs (et al , and 1997, Le Tissier et al , et al ). Three subgroups of PoERV A, B and C are recognised based on interference properties which by definition indicates that these viruses use separate receptors to enter cells. Both subgroup A and B have infect a range of human cell types (Le Tissier et al ) but with an efficiency about 10² to 10³ lower the for amphotropic murine leukaemia virus (Onions et al , Takeuchi et al ) which is widely used as the basis of human gene therapy vectors. Subgroup C has only been shown to infect one human cell line, HT-1080-1 cells, and it is not yet clear whether this virus has a general ability to infect human cells (Takeuchi et al ). A further feature limiting the spread of these viruses in vitro is that on infection the proviruses frequently become transcriptionally silent. 4.2.2 The sequence of the PK15 virus The complete nucleotide sequence of two PoERVs has been obtained (and 1997, Akiyoshi et al ). In addition envelope sequences of other viruses have been obtained (Banerjee et al ). Some of these may represent type variants within subgroups A, B and C but many have yet to be analysed by pseudotype analysis to confirm their subgroup status. PoERV-B1 and C share identity within gag and identity within pol, and are closely related to the gibbon ape leukaemia virus (GaLV). The primer binding sites have been used as a basis for classifying retroviruses. Surprisingly, PoEV-B1 uses a tRNA binding site complementary to glycine 2, a feature which has not been recorded for any other retrovirus, whereas PoERV-C utilises a proline tRNA primer. The Long terminal repeat of the PoERV-B1 virus contains enhancers within the U3 region that has motifs that indicative of expression in lymphoid or haematopoietic cells. These include LyF-l (Lo et al ) E47 ( Holenberg et al ) and ETS-1 . It is of interest that spontaneous expression of PoERVs has been observed from activated primary lymphocytes and from endothelial cells (et al , et al ) 4.2.4Relevance of the data for the development of porcine xenotransplantation The acquisition of the complete PoERV sequence has enabled a number of tools to be developed for the detection and analysis of the expression of PoERVs in porcine and human tissue. The development of clinical trails using porcine tissue must exclude the possibility that infection of human cells will occur with subsequent production of mutated or recombinant proviruses that may spread through the human population (Stoye and Coffin 1995). The poor infectivity and limited expression of PoERV in vitro indicates that in vivo the virus would probably be unable to establish a productive infection. However, confounding factors are the effects of iatrogenic immunosuppression and the probable loss of the protective complement dependent lysis of retroviruses in pigs transgenic for human complement regulatory genes. Consequently whole organ xenografts may pose a higher risk than tissue transplants like islet cells since, immunosuppression is required, the tissue is likely to be transgenic for complement regulatory genes and a wider range of tissues including endothelial cells are transplanted. A decision tree for progressing to clinical trials might encompass several elements: 1.Expression of the virus in the pigs: Are proviruses present in every pig and are they expressed? All pigs examined thus far appear to contain multiple copies of PoERV A & B (Le Tissier et al ) whereas subgroup C is more plymorphic with some pigs lacking a prototype subgroup C env sequnce with others having 50 copies (Onions et al ). Expression of PoERV sequences appears to be common in many tissues but may be restricted to certain cells like endothelial cells or lymphocytes (G. pers comm.). However recent evidence indicates that some pigs have low level viraemiias (and Galbraith pers comm). 2.Analysis of primate models with porcine xenotransplants. Provided the cells of the primates can be shown to be permissive for infection by PoERV they are potentially valuable models for assessing the safety of porcine xenotransplantation. Provided the confounding factor of micro-chimaerism can be controlled it should be possible to determin if PoERV infects the primate tissues. In the event that PoERV genomes can be identified in primate cells great caution would be needed before considering clinical trials of whole organ transplants. 3.Analysis of patients who have received porcine xenografts or xenotransplants. There are a number of patients who have already received porcine islet cell xenografts that have been examined for PoERV and no evidence for infection has been observed (Heneine et al ). One form of analysis is to determine if PoERV sequences are present in PBMCs of these patients. This is more complex than it appears as microchimaerism from porcine cells present in peripheral blood can complicate the analysis. One approach around this problem is to use quantitative PCR techniques and to compare PoERV copy number to a reference porcine gene sequence. Ratios of PoERV copy number to control gene copy number outside a defined range would be indicative infection. However, analysis of natural models of oncovirus infection indicates that a broader range of assays is required to define all infection states. For instance in cats infected with FeLV the majority eliminate the infection and develop antibodies to the virus. This contrast with HIV and FIV where antibodies indicate infection. Persistently infected animals, which develop disease and transmit the virus, exhibit plasma viraemias and plasma antigenaemia. However other infection states exist. In one the virus is sequestered in epithelial sites. These animals usually lack virus or viral genomes in the circulation but may have viral antigen and possibly anti-viral antibody. In a true latent state FeLV genomes are detectable in the bone marrow and to a variable extent in the peripheral blood. Such animals usually have detectable anti-viral antibody but no viral antigen in the plasma. Consequently one should be cautious in using one assay system to define the viral status of patients. Western blot systems using both purified virions and recombinant viral proteins have been validated for screening for human antibody to PoERV (Galbraith and Onions unpublished) and highly and RT- PCR systems have been developed to screen for plasma viraemias (Lovatt et al submitted for publication, Lovatt and Onions unpublished). 4.2.5 The future of porcine xenotransplantation With the caveats expressed above it may be possible to proceed in a cautious, step by step approach, to clinical trails, in which a rigorous analysis of the recipient patients will play an important part in validating the safety of the procedure. Should the risks of the endogenous retrovirus pose an unacceptable level of risk there are longer term solutions. Vaccination of patients before transplantation is not unrealistic as there are effective vaccines available against gammaretroviruses in contrast to the lentiviruses. 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Scientific Progress and Public Safety: Xenotransplantation Clinical Trials in the , MD, MSPH Centers for Disease Control and Prevention, , Report for 1999 consultation In , the first statement of proposed US policy on xenotransplantation was published for public comment. () Draft Guideline on Infectious Disease Issues in Xenotransplantation, was developed jointly by four PHS agencies of (), which is the US Governments principal agency for protecting the health of all Americans. The four PHS agencies include the Centers for Disease Control and Prevention [CDC], the emergency response / preventive medicine agency; the Food and [FDA], the regulatory agency for food, drugs and devices; [NIH], the biomedical research agency; and the and [HRSA], the agency responsible for health care programs and health professions work force. In January 1997, the Office of the Secretary of () organized an on Xenotransplantation. is chaired by the Deputy Assistant for Science Policy, DHHS and has representatives from CDC, NIH, FDA, HRSA, and the Office of General , DHHS. is charged with recommending to the Secretary an integrated public policy on xenotransplantation, and is responsible for developing a revised DHHS Guideline on Infectious Disease Issues in Xenotransplantation, anticipated to be published in 1999. Evolving public policy in the US includes a number of safeguards intended to maximize the safety of the public as xenotransplantation moves from laboratory research into clinical trials. The draft PHS Guideline on Infectious Disease Issues in Xenotransplantation, published in the Federal Register in , and the revised Guideline, anticipated to be published in 1999, discuss general principles for the prevention and control of infectious diseases that may be associated with xenotransplantation, and provides guidance to sponsors of xenotransplantation clinical trials about the development and implementation of clinical protocols. All xenotransplantation clinical trials conducted within the are subject to regulation by the FDA. FDA regulations specify that any protocol that does not adequately address the safety issues and risks posed by the xenograft is subject to clinical hold (meaning that the trial can not proceed). For example, in response to research findings reported in 1997 by and colleagues and and colleagues in the , and by and colleagues at the FDA in the , the FDA placed all porcine xenotransplantation clinical trials on hold on October 16, 1997, pending development by sponsors of sensitive and specific assays for 1) detection of infectious porcine endogenous retrovirus (PERV) in porcine xenografts, 2) post- transplant surveillance for PERV infections in porcine xenograft recipients, as well as the development of informed consent documents that explain the potential clinical implications of the capacity of PERV to infect human cells in vitro. This hold is lifted on a protocol-by- protocol basis as individual sponsors meet these requirements. In addition to regulatory oversight provided by the FDA, () also has a regulatory role vis-à-vis xenotransplantation: it regulates the importation of all animals and animal-origin materials that could represent a disease risk to US livestock, as well as the interstate transport of animals or animal-origin materials within the US. The CDC, Division of Quarantine, regulates the importation of certain animals, including nonhuman primates, because of their potential to cause serious outbreaks of communicable disease in humans. Formal advisory committees provide a mechanism whereby the U.S. government can seek advice on policy development from non- government experts. () is being developed by DHHS to address challenges raised by the potential impact of xenotransplantation on society as a whole. As currently envisioned, the SACX will consider the full range of issues raised by xenotransplantation, including ongoing and proposed protocols, and make recommendations to the Secretary on policy and procedures. The SACX will also provide a forum for public discussion of scientific, , public health, ethical, legal and social issues. The PHS recognizes that substantial concerns exist within both scientific and lay communities that nonhuman primate xenografts may be associated with a greater risk of introducing xenogeneic infections. Existing scientific data is inadequate to clearly address this concern. The SACX would provide a forum in which, for example, issues raised by the use of nonhuman primate xenografts could be addressed and recommendations developed on whether and under what conditions the use of nonhuman primate xenografts would be appropriate in the . Another advisory mechanism for xenotransplantation is the Xenotransplantation Subcommittee of which was specifically established to provide guidance to FDA on xenotransplantation policy. CDC has utilized as a source of expert guidance. A pilot is under development by the PHS. It is envisioned that this pilot will be expanded into a National Database to collect data from all clinical centers conducting trials in xenotransplantation and all animal facilities providing animals or xenografts for clinical use. Such a database would increase the capability to recognize rare events that may represent consequences of xenogeneic infections, and to link, on a national level, these events to exposures. Sponsors of xenotransplantation clinical trials are also required to archive biologic specimens from the source animals, xenograft products, and xenograft recipients for 50 years. These specimens are to be reserved for testing should it become necessary to conduct CDC-directed PHS investigations of xenotransplantation associated adverse events. As such, they represent a safeguard, facilitating effective emergency response to public health threats that may be triggered by a xenotransplant. The option of developing a central publicly funded PHS biologic specimen archive is under consideration. Federal law protects the right of citizens to access information about developing policy. The first amendment to the guarantees the right of citizens to petition the government for redress of grievances. The DHHS is currently reviewing a petition on xenotransplantation policy received from a group of concerned citizens. The ability of citizens to influence policy development is further protected by the 1966 Freedom of Information Act, which guarantees citizens access to government documents, and the 1976 Government in Sunshine Act, which prohibits the government from working in secrecy. The PHS Guideline on Infectious Disease Issues in The Revised PHS Guideline emphasizes the importance of the protocol development and review process, the informed consent and education processes, and development of adequate protocols for pre-transplant screening and post-transplant surveillance for infectious diseases. Protocol Development and Review The xenotransplantation team should include (1) infectious disease physician(s) with expertise in zoonoses, transplantation, and epidemiology; (2) veterinarian(s) with expertise in infectious diseases of the source animal; (3) specialist(s) in hospital epidemiology and infection control; and (4) experts in research and diagnostic microbiology laboratory methodologies as participants. Clinical xenotransplantation centers should have expertise with comparable allotransplantation procedures, utilize accredited virology and microbiology laboratories, and have the capability to identify human and animal infections agents using in vitro and in vivo methodologies (on site or through collaborations). Investigators must submit an Investigational New Drug (IND) application for FDA review and authorization before proceeding with xenotransplantation clinical trials. Responsibilities for the design and conduct of xenotransplantation clinical trials rests with the sponsor of the FDA IND.