Infectious disease risks in xenotransplantation

1 DETERMINANTS OF INFECTIOUS RISK IN TRANSPLANTATION

Xenotransplantation, the transplantation of viable cells, organs, or tissues between species, is proposed as a solution to the shortage of human organs for the treatment of organ failure. Recent advances in the management of immunological and metabolic barriers to pig-to-primate xenotransplantation improve the likelihood that clinical trials of xenotransplantation are feasible. The risk of infection in xenotransplantation due to organisms derived from nonhuman source animal species remains an important, and ill-defined, challenge to clinical xenotransplantation.

As for all hosts, the risk for infection in the recipient after transplantation reflects the interaction between 2 factors: the epidemiology of infection including the dose, intensity, and virulence of the specific organisms present in the recipient and the graft; and the “net state of immunosuppression”—a conceptual measure of any factors that may contribute to the risk for active infection, such as the intensity and duration of immunosuppression.1, 2 The transmission of infection with viable cells and tissues is efficient, notably for intracellular organisms and latent viruses, and is enhanced by the immunosuppression required to prevent graft rejection. Some of the earliest discussions of unexpected, donor-derived infections in transplantation surrounded xenotransplantation.3-5 In human allotransplantation, donor screening is used as a basis for decisions regarding donor suitability and for prophylactic or therapeutic interventions in recipients. It is understood that a risk for disease transmission exists in all forms of transplantation. Some common infectious transmissions are considered “acceptable” based on the availability of prophylactic therapies, including cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella zoster virus (VZV), and recently, hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV (under institutional review board–approved protocols). Reported, unexpected, donor-derived infections occur in approximately 0.2% of transplants in the United States including organisms undetected in donor screening.6 Based on the limited time available to procure and allocate organs from deceased donors, screening data from organ donors available prior to transplantation is generally limited to a panel of serologic and molecular tests; data from blood and urine (and bronchoscopic samples from lung donors) cultures (potentially while receiving antimicrobial therapy) are delayed. Any specialized tests (eg, Strongyloides serologies) are not available until after the organs have been implanted. Despite these limitations, the rate of transmission of infection from human organ donors is very low.

The evaluation and exclusion of potential organ donors is considered in the context of the shortage of organs for transplantation. Organs are increasingly used from donors considered at “Increased Risk for the Transmission of Infectious Disease” under US Public Health Service (PHS) Guidelines targeting transmission of HIV, HBV, and HCV.7 Microbiologic testing of donors including serologic and nucleic acid testing (NAT) and effective treatment options for HIV, HCV, and HBV have allowed use of “PHS increased risk donor” organs.2, 4 Transplant programs utilizing “increased risk” organs are required by the Centers for Medicare & Medicaid Services (CMS) Guidelines and Organ Procurement and Transplant Network (OPTN) Policies to obtain informed consent from the patient prior to transplantation and to offer follow-up testing for HIV, HCV, and HBV. Organ donation from increased risk donors has resulted in few transmission events—the exception being potential donors with negative serologic and molecular viral assays for HIV, HCV, or HBV, likely in the “window period” immediately following viral infection.8 The individual with organ failure may interpret the degree of “risk” with informed consent differently than public health authorities, infectious disease specialists, and transplant physicians and surgeons.

2 INFECTION IN XENOTRANSPLANTATION

The terms “xenosis,” “direct zoonosis,” and “xenozoonosis” were coined to reflect the unique epidemiology of infection due to organisms potentially carried by nonhuman tissues. Experience with immunocompromised individuals suggests that certain types of pathogens are likely to emerge after transplantation (reviewed in1, 5, 9, 10) (Table 1). With transplant immunosuppression, both common pathogens of transplant recipients and unique porcine pathogens may emerge. Studies of xenotransplantation recipients and humans with exposure to pigs or pig products have not revealed any consistent patterns of infection. Early preclinical studies focused on the risk of infection when using miniature swine as xenograft donors for immunosuppressed nonhuman primates. Swine were found to be colonized by Actinomyces sp. and Streptococcus suis (J. Fishman, unpublished data) but otherwise had readily identified bacteria as the etiology of line-related bacteremia, pneumonia, or infections related to surgical procedures. The rate of antimicrobial-resistant infection in immunosuppressed recipients of porcine allografts and xenografts (baboons) was reduced through minimization of routine antimicrobial (first-generation cephalosporin surgical perioperative) prophylaxis in both the donor herd and in recipients. Isolation of resistant (fluoroquinolone and cephalosporin) bacteria is reduced by careful attention to routine antibiotic use in pig rearing. Intravenous ganciclovir was used for posttransplant prophylaxis against both porcine cytomegalovirus (PCMV) and baboon cytomegalovirus (BCMV).11, 12

3 SCREENING OF DONOR SWINE

As for deceased donor screening, the goal of animal husbandry for xenotransplantation is to exclude potential pathogens for the human recipient and to prevent the introduction of new pathogens into breeding colonies from the environment and by care providers. It seems likely that the way in which the level of safety is achieved need not be uniform so long as the transplanted tissues do not pose a microbiologic hazard to the recipient. A potential benefit of xenotransplantation is that the screening paradigm can be more intensive than that used for human organ donors, limited only by the availability of microbiologic assays. Assays may be applied to an isolated herd of donor animals and/or to individual source animals. Validated assays for organisms in swine are largely limited to serologic testing, microscopic examination (stool, parasites), and standard microbiologic cultures for common pathogens, available in a small number of commercial or veterinary laboratories. Other assays (eg, nucleic acid testing for viruses) are more limited. Microbiological standards for pig husbandry should be considered “dynamic”—subject to revision based on experimental and clinical data and reflecting the evolution of testing strategies.

Based on experience, 2 separate lists of organisms may facilitate development of swine for xenotransplantation. One group of pathogens is excluded from breeding herds as a measure of pig health status. This list is guided by standard veterinary practice, including routine vaccines with the addition of microbially restricted and mammalian protein-free diets, filtered water, and special housing. Swine for xenotransplantation can be bred in “biosecure facilities” to prevent introduction of potential pig or human pathogens, isolated from other swine and animals, including rodents and birds, often with care providers gowned and gloved. Routine antimicrobial use should be restricted. It is not generally considered useful to develop gnotobiotic swine, which grow less well than swine with normal microbial complements. Genetic modification of swine to reduce graft rejection or for the elimination of endogenous retroviruses necessitates transgenic methods with nuclear transfer in sterile environments and subsequent embryo transfer to surrogate gilts.13-17

A second list of potential swine-derived organisms was created to identify potential pathogens based on experience with immunosuppressed human allotransplant recipients and pig-to-primate xenotransplantation (Table 2). Exclusion, to the degree possible, of these potential pathogens was termed “designated pathogen free” status.5, 9, 10, 18, 19 Pig-derived infections in immunosuppressed humans have not been reported with the possible exception of hepatitis E virus (HEV).

4 THE IMPACT OF VIRAL INFECTION AFTER XENOTRANSPLANTATION

Viral infection is common after organ transplantation due to the efficient transmission of viruses with viable cells and suppression of T lymphocyte–mediated antiviral immune responses in the MHC-disparate graft. Multiple factors increase viral replication subsequently including immunosuppression, graft rejection, proinflammatory cytokines, and ischemia-reperfusion injury. In xenotransplantation, these factors may also contribute to replication of porcine endogenous retrovirus (PERV), porcine cytomegalovirus (PCMV) and porcine lymphotropic herpesvirus (PLHV-1, -2, -3). Additional infections observed in immunosuppressed swine, as well as normal animals, include porcine circovirus (PCV) and adenovirus. The diagnosis of infections due to these common porcine organisms in primate recipients has been addressed by the development of sensitive, quantitative NAT assays for PERV, PLHV, PCMV, and PCV.

Each virus is associated with a specific clinical syndrome in swine and in nonhuman primate xenograft recipients. PCMV causes infection restricted to the porcine renal xenografts with endothelial activation, consumptive coagulopathy, and early graft loss.12, 20-23 PCMV can be excluded from pig colonies by early weaning and careful isolation.24-27 PLHV has been associated with a form of posttransplantation lymphoproliferative disorder (PTLD) in immunosuppressed swine undergoing stem cell transplantation.28, 29 Porcine herpesviruses appear to be relatively species-specific; active graft infection is not associated with systemic infection in primate recipients of xenografts, although shed virus may be detected in circulation.27, 30 Other viral infections tend to be self-limited in normal swine. Porcine circovirus type 2 (PCV2), for example, may cause pneumonia and wasting syndrome and depresses various innate immune functions.31, 32 Transmission of these viruses to human hosts has not been reported.

5 PORCINE ENDOGENOUS RETROVIRUS (PERV)

The isolation and sequencing of PERV was based on early studies of a retrovirus associated with swine lymphoma.33-42 Concern about retroviral transmission in xenotransplantation relates to the potential for “silent” transmission or asymptomatic infection with insertional effects and altered gene regulation or oncogenesis. Three closely related C-type porcine endogenous retroviruses (PERV A, B, C) have been identified in swine that possess infectious potential.37, 40, 43-46 Human cellular receptors for PERV-A, Human porcine endogenous retrovirus receptor (HuPAR)-1 and HuPAR-2, have been identified.43 PERV-A and -B, can infect human and pig cells in vitro, the third sub-group, PERV-C, infects only pig cells.37, 39, 40, 43, 44, 46-50Full-length proviral copy numbers of PERV-A/B among domestic pig genomes range between 7 and 14 copies; not all carry infectious potential.40, 51 The proviral copy numbers increase with inbreeding and with cultivation for nuclear transfer.52 Exogenous recombinant viruses containing the receptor-binding site of PERV-A and segments of PERV-C (PERV AC) demonstrate increased replication efficiency in vitro and may initiate an autoinfection cycle with genomic AC recombinants.44, 53, 54, 48 Within inbred herds of swine, individual animals may be characterized as PERV transmitter or nontransmitters.55 These characteristics are not stable.

PERV mRNAs are expressed in all pig tissues and in all breeds of swine tested to date.40, 56-59 There is variation between tissues in terms of the size and quantity of PERV mRNA transcripts, consistent with in vivo recombination and/or processing.40, 60, 61 Some swine lack PERV-C or carry PERV-C that is lacking a locus potentially associated with PERV recombination and virus transmission to human target cells.62 Such isolated pig herds lacking PERV-C have been utilized in clinical trials of porcine islet xenotransplantation.63-65 PERV infects some nonhuman primate cells in vitro but does not replicate in these cells, making primate studies less informative regarding PERV infectious risks.66, 67

Highly sensitive and specific quantitative molecular methods have been developed to characterize PERV infections; validated assays for use in human subjects do not exist (Table 1). In vitro systems for infectious virus utilize coculture of porcine cells with adenovirus-5-transformed human embryonic kidney target cells (HEK293), which do not express the endogenous restriction factor APOBEC and are permissive to PERV infection.37, 50, 68 Three groups of primary human hepatocytes were not infectable by PERV.69 Human PBMCs were infectable with PERV in vitro only with high titers of highly infectious virus containing numerous repeats in the LTR (long terminal repeat elements).70 Evidence of infection of human cells by porcine endogenous retrovirus after islet xenotransplantation in SCID (severe combined immunodeficiency disorder) mice was found to be erroneous and an artifact of pseudotyping of PERV by murine leukemia virus (MLV).60, 71, 72 Most primary human cells have not been infectable by PERV; human vascular endothelial cells (HUVEC) and peripheral blood mononuclear cells are infectable by certain strains of PERV in vitro.39, 47, 49, 50, 73-75 Some studies utilize “human cells” as targets for infection using the HEK293T cell line, which lacks certain innate restriction mechanisms. Human APOBEC3 and other cellular restriction factors may inhibit PERV replication.68, 76 Further studies are needed to define the susceptibility of primary human cells to PERV infection and any mechanisms serving to limit productive infection despite the presence of functional PERV receptors.75

To date, all preclinical and clinical xenotransplantation studies using pig cells, tissues, and organs have failed to demonstrate transmission of PERV to humans including transplantation of porcine pancreatic islets and over 200 individuals exposed to pig cells or tissues or ex vivo perfusion of pig organs or pig cell–based bioreactors.64, 65 In these subjects, no evidence for virus transmission was obtained and no antibodies against PERV or provirus integration in blood cells of the patients were observed. In one early study, persistent microchimerism was observed in 23 patients for up to 8.5 years after treatment without PERV transmission.77 If human infection should occur, PERV is susceptible in vitro to nucleoside and non-nucleoside reverse transcriptase inhibitors in common clinical use.49, 78-83

Theoretical strategies have been developed to prevent PERV transmission including use of PERV-C-negative or low virus producing pigs, vaccination, antiretroviral therapy, RNA interference therapies, and PERV knockout animals using Clustered Regularly Interspaced Short Palindromic Repeats-Caspase9 (CRISPR-Cas9) techniques.84, 85 Using CRISPR-Cas9 to target the polymerase gene of PERV elements, inactivation was achieved of all 62 copies of PERV in the immortalized porcine kidney epithelial cell line PK15, which normally releases high levels of infectious PERV in vitro.15 All PERV elements were mutated, and viral replication and reverse transcriptase activity were no longer detected in vitro; transmission to human cells in co-culture studies was no longer demonstrable.15 PERV-C-negative pig fibroblasts were similarly treated for use in somatic cell nuclear transfer to generate PERV-inactivated embryos, carried by PERV-C-negative surrogate sows.86 Although the infectivity status of these fibroblasts at baseline was not presented, PERV inactivation (~25 copies) was confirmed at the DNA and RNA levels; further studies are underway. Xenografts from these animals have not yet been reported. Concerns regarding off-site genomic modifications by CRISPR technology are under investigation.87

6 MICROBIOLOGIC SAFETY IN CLINICAL TRIALS OF XENOTRANSPLANTATION

7 UNKNOWN PATHOGENS AND THE FUTURE

In xenotransplantation, potential infectious risks pose some unique challenges. Despite significant advances, the absolute risk for infections in xenotransplantation remains unknown in the absence of human studies. Regulatory guidance regarding the production of source animals and surveillance in recipients will require adjustment as clinical data emerge. New microbiological assays will be required to screen swine for potential human pathogens and for the diagnosis of pig-specific pathogens in humans. Such assays will take advantage of archived samples should infectious syndromes emerge. The risk for infection will depend on factors including the nature and intensity of immunosuppression and the quantity and “virulence” of any organisms for human cells including functional receptors and intrinsic restriction systems. The broad spread of infection from xenograft recipients to the community seems unlikely; an ongoing assessment is required to detect any transmission events that may occur.93, 94 A cautious approach seems reasonable. The opportunity to expand organ availability for transplantation for patients with organ failure merits careful consideration.

DISCLOSURE

The author of this manuscript has conflicts of interest to disclose as described by the American Journal of Transplantation. Dr. Fishman is a consultant for infection in xenotransplantation to Revivicor, United Therapeutics, Xenotherapeutics, Harvard Medical School, University of Alabama, Columbia University, and the World Health Organization.