|
1
|
Stewart AG, Fishman JA. Surveillance and prevention of infection in clinical xenotransplantation. Clin Microbiol Rev 2025; 38:e0015023. [PMID: 39887237 PMCID: PMC11905366 DOI: 10.1128/cmr.00150-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2025] Open
Abstract
SUMMARYXenotransplantation, the transplantation of living organs, tissues, or cells between species, carries the potential to address the global shortage of human organs for patients with end-stage organ failure. Recent advances in genetic engineering have improved prospects for clinical xenotransplantation by reducing immune and inflammatory responses to grafts, controlling coagulation on endothelial surfaces, and modifying viral risks, including the porcine endogenous retrovirus (PERV). Management of infectious risks posed by clinical xenotransplantation requires meticulous attention to the biosecure breeding and microbiological surveillance of source animals and recipients and consideration of novel infection control requirements. Infectious risks in xenotransplantation stem from both known human pathogens in immunosuppressed transplant recipients and from porcine organisms for which the clinical manifestations, microbial assays, and therapies are generally limited. Both known and unknown zoonoses may be transmitted from pigs to humans. Some pig-specific pathogens do not infect human cells but have systemic manifestations when active within the xenograft, including porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV), which contributes to graft rejection and consumptive coagulopathy. The role of porcine endogenous retrovirus (PERV) in humans remains uncertain despite the absence of documented transmissions and the availability of swine with inactivated genomic PERV. New technologies, such as metagenomic sequencing and multi-omics approaches, will be essential for detection of novel infections and for understanding interactions between the xenograft, the host's immune system, and potential pathogens. These approaches will allow development of infection control protocols, pathogen surveillance requirements, and tailored antimicrobial therapies to enhance the safety and success of clinical xenotransplantation.
Collapse
Affiliation(s)
- Adam G. Stewart
- Transplant Infectious Disease and Compromised Host Program, MGH Transplant Center, Harvard Medical School, Boston, Massachusetts, USA
| | - Jay A. Fishman
- Transplant Infectious Disease and Compromised Host Program, MGH Transplant Center, Harvard Medical School, Boston, Massachusetts, USA
| |
Collapse
|
|
2
|
Song YW, Pan ZQ. Reducing porcine corneal graft rejection, with an emphasis on porcine endogenous retrovirus transmission safety: a review. Int J Ophthalmol 2019; 12:324-332. [PMID: 30809491 DOI: 10.18240/ijo.2019.02.21] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2018] [Accepted: 11/28/2018] [Indexed: 01/08/2023] Open
Abstract
Donor cornea shortage is a primary hurdle in the development of corneal transplantation. Of all species, porcine corneas are the ideal transplantation material for humans. However, the xenoimmune rejection induced by porcine corneal xenotransplantation compromises surgical efficacy. Although the binding of IgM/IgG in human serum to a genetically modified porcine cornea is significantly weaker than that of the wild type (WT), genetically modified porcine corneas do not display a prolonged graft survival time in vivo. Conversely, costimulatory blockade drugs, such as anti-CD40 antibodies, can reduce the xenoimmune response and prolong graft survival time in animal experiments. Moreover, porcine endothelial grafts can survive for more than 6mo with only the subconjunctival injection of a steroid-based immunosuppressants regime; therefore, they show great value for treating corneal endothelial disease. In addition, zoonotic transmission is a primary concern of xenotransplantation. Porcine endogenous retrovirus (PERV) is the most significant virus assessed by ophthalmologists. PERV integrates into the porcine genome and infects human cells in vitro. Fortunately, no evidence from in vivo studies has yet shown that PERV can be transmitted to hosts.
Collapse
Affiliation(s)
- Yao-Wen Song
- Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Science Key Laboratory, Beijing 100730, China
| | - Zhi-Qiang Pan
- Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology and Visual Science Key Laboratory, Beijing 100730, China
| |
Collapse
|
|
3
|
Scobie L, Crossan C, Mourad NI, Galli C, Perota A, Gianello P. Viral pathogens: What are they and do they matter? Xenotransplantation 2018; 25:e12412. [PMID: 29913035 DOI: 10.1111/xen.12412] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Revised: 04/24/2018] [Accepted: 05/02/2018] [Indexed: 02/06/2023]
Affiliation(s)
- Linda Scobie
- Department of Biological and Biomedical Sciences, School of Health and Life Sciences, Glasgow Caledonian University, Glasgow, UK
| | - Claire Crossan
- Department of Biological and Biomedical Sciences, School of Health and Life Sciences, Glasgow Caledonian University, Glasgow, UK
| | - Nizar I Mourad
- Laboratoire de Chirurgie Expérimentale et Transplantation, Université Catholique de Louvain, Brussels, Belgium
| | | | | | - Pierre Gianello
- Laboratoire de Chirurgie Expérimentale et Transplantation, Université Catholique de Louvain, Brussels, Belgium
| |
Collapse
|
|
4
|
Liu Z, Hu W, He T, Dai Y, Hara H, Bottino R, Cooper DKC, Cai Z, Mou L. Pig-to-Primate Islet Xenotransplantation: Past, Present, and Future. Cell Transplant 2017; 26:925-947. [PMID: 28155815 PMCID: PMC5657750 DOI: 10.3727/096368917x694859] [Citation(s) in RCA: 46] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2016] [Accepted: 03/21/2017] [Indexed: 12/17/2022] Open
Abstract
Islet allotransplantation results in increasing success in treating type 1 diabetes, but the shortage of deceased human donor pancreata limits progress. Islet xenotransplantation, using pigs as a source of islets, is a promising approach to overcome this limitation. The greatest obstacle is the primate immune/inflammatory response to the porcine (pig) islets, which may take the form of rapid early graft rejection (the instant blood-mediated inflammatory reaction) or T-cell-mediated rejection. These problems are being resolved by the genetic engineering of the source pigs combined with improved immunosuppressive therapy. The results of pig-to-diabetic nonhuman primate islet xenotransplantation are steadily improving, with insulin independence being achieved for periods >1 year. An alternative approach is to isolate islets within a micro- or macroencapsulation device aimed at protecting them from the human recipient's immune response. Clinical trials using this approach are currently underway. This review focuses on the major aspects of pig-to-primate islet xenotransplantation and its potential for treatment of type 1 diabetes.
Collapse
Affiliation(s)
- Zhengzhao Liu
- Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, P.R. China
| | - Wenbao Hu
- Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, P.R. China
| | - Tian He
- Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, P.R. China
| | - Yifan Dai
- Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing, Jiangsu, P.R. China
| | - Hidetaka Hara
- Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, Birmingham, AL, USA
| | - Rita Bottino
- Institute for Cellular Therapeutics, Allegheny-Singer Research Institute, Pittsburgh, PA, USA
| | - David K. C. Cooper
- Xenotransplantation Program/Department of Surgery, The University of Alabama at Birmingham, Birmingham, AL, USA
| | - Zhiming Cai
- Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, P.R. China
| | - Lisha Mou
- Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong, P.R. China
| |
Collapse
|
|
5
|
Abstract
PURPOSE OF REVIEW Diabetes is medical and social burden affecting millions around the world. Despite intensive therapy, insulin fails to maintain adequate glucose homeostasis and often results in episodes of hypoglycemic unawareness. Islet transplantation is a propitious replacement therapy, and incremental improvements in islet isolation and immunosuppressive drugs have made this procedure a feasible option. Shortage of donors, graft loss, and toxic immunosuppressive agents are few of many hurdles against making human allogenic islet transplantation a routine procedure. RECENT FINDINGS Xenografts-especially pig islets-offer a logical alternative source for islets. Current preclinical studies have revealed problems such as optimal islet source, zoonosis, and immune rejection. These issues are slowing clinical application. Genetically modified pigs, encapsulation devices, and new immune-suppressive regimens can confer graft protection. In addition, extrahepatic transplant sites are showing promising results. Notwithstanding few approved clinical human trials, and available data from non-human primates, recent reports indicate that porcine islets are closer to be the promising solution to cure diabetes.
Collapse
Affiliation(s)
- Bassem F Salama
- Department of Surgery, University of Alberta, 5.002 Li Ka Shing Bldg, 8602 112 Street, Edmonton, AB, T6G 2E1, Canada
- Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada
| | - Gregory S Korbutt
- Department of Surgery, University of Alberta, 5.002 Li Ka Shing Bldg, 8602 112 Street, Edmonton, AB, T6G 2E1, Canada.
- Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.
| |
Collapse
|
|
6
|
Mathis AS, Egloff G, Ghin HL. Calcineurin inhibitor sparing strategies in renal transplantation, part one: Late sparing strategies. World J Transplant 2014; 4:57-80. [PMID: 25032096 PMCID: PMC4094953 DOI: 10.5500/wjt.v4.i2.57] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/08/2014] [Revised: 03/25/2014] [Accepted: 05/14/2014] [Indexed: 02/05/2023] Open
Abstract
Kidney transplantation improves quality of life and reduces the risk of mortality. A majority of the success of kidney transplantation is attributable to the calcineurin inhibitors (CNIs), cyclosporine and tacrolimus, and their ability to reduce acute rejection rates. However, long-term graft survival rates have not improved over time, and although controversial, evidence does suggest a role of chronic CNI toxicity in this failure to improve outcomes. Consequently, there is interest in reducing or removing CNIs from immunosuppressive regimens in an attempt to improve outcomes. Several strategies exist to spare calcineurin inhibitors, including use of agents such as mycophenolate mofetil (MMF), mycophenolate sodium (MPS), sirolimus, everolimus or belatacept to facilitate late calcineurin inhibitor withdrawal, beyond 6 mo post-transplant; or using these agents to plan early withdrawal within 6 mo; or to avoid the CNIs all together using CNI-free regimens. Although numerous reviews have been written on this topic, practice varies significantly between centers. This review organizes the data based on patient characteristics (i.e., the baseline immunosuppressive regimen) as a means to aid the practicing clinician in caring for their patients, by matching up their situation with the relevant literature. The current review, the first in a series of two, examines the potential of immunosuppressive agents to facilitate late CNI withdrawal beyond 6 mo post-transplant, and has demonstrated that the strongest evidence resides with MMF/MPS. MMF or MPS can be successfully introduced/maintained to facilitate late CNI withdrawal and improve renal function in the setting of graft deterioration, albeit with an increased risk of acute rejection and infection. Additional benefits may include improved blood pressure, lipid profile and serum glucose. Sirolimus has less data directly comparing CNI withdrawal to an active CNI-containing regimen, but modest improvement in short-term renal function is possible, with an increased risk of proteinuria, especially in the setting of baseline renal dysfunction and/or proteinuria. Renal outcomes may be improved when sirolimus is used in combination with MMF. Although data with everolimus is less robust, results appear similar to those observed with sirolimus.
Collapse
|
|
7
|
Porcine endogenous retroviruses in xenotransplantation--molecular aspects. Viruses 2014; 6:2062-83. [PMID: 24828841 PMCID: PMC4036542 DOI: 10.3390/v6052062] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2014] [Revised: 04/15/2014] [Accepted: 04/26/2014] [Indexed: 02/06/2023] Open
Abstract
In the context of the shortage of organs and other tissues for use in human transplantation, xenotransplantation procedures with material taken from pigs have come under increased consideration. However, there are unclear consequences of the potential transmission of porcine pathogens to humans. Of particular concern are porcine endogenous retroviruses (PERVs). Three subtypes of PERV have been identified, of which PERV-A and PERV-B have the ability to infect human cells in vitro. The PERV-C subtype does not show this ability but recombinant PERV-A/C forms have demonstrated infectivity in human cells. In view of the risk presented by these observations, the International Xenotransplantation Association recently indicated the existence of four strategies to prevent transmission of PERVs. This article focuses on the molecular aspects of PERV infection in xenotransplantation and reviews the techniques available for the detection of PERV DNA, RNA, reverse transcriptase activity and proteins, and anti-PERV antibodies to enable carrying out these recommendations. These methods could be used to evaluate the risk of PERV transmission in human recipients, enhance the effectiveness and reliability of monitoring procedures, and stimulate discussion on the development of improved, more sensitive methods for the detection of PERVs in the future.
Collapse
|
|
8
|
Gola J, Mazurek U. Detection of porcine endogenous retrovirus in xenotransplantation. Reprod Biol 2014; 14:68-73. [PMID: 24607257 DOI: 10.1016/j.repbio.2014.01.006] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2013] [Revised: 01/22/2014] [Accepted: 01/23/2014] [Indexed: 10/25/2022]
Abstract
Xenotransplantation can provide a virtually limitless supply of cells, tissues and organs for a variety of therapeutic procedures. Cells and tissues for use in human transplantation procedures could be supplied using material taken from pigs. However, there is a potential risk of transmission of porcine infectious agents, including porcine endogenous retroviruses (PERVs), to a novel human host, with as yet unknown consequences. Three subtypes of PERV have been identified, of which both PERV-A and PERV-B have the ability to infect human cells in vitro. The third subtype, PERV-C, does not show this ability. Recombinant PERV-A/C forms have demonstrated infectivity in human cell culture. Monitoring in xenotransplantation should comprise screening of the source pig herd (PERV-A and PERV-B level expression assessment, PERV-C detection) and screening of recipients (differentiation between PERV transmission and chimerism). The detection of PERVs includes analyses of both DNA and RNA (PCR and RT-PCR), quantitative determination of the level of PERV nucleic acids (real-time PCR and real-time RT-PCR), assessment of reverse transcriptase (RT) activity (RT assays) and viral and recipient protein detection (immunological methods). In summary, all available methods should be used in monitoring of PERVs in xenotransplantation, and caution should be exercised at all stages of monitoring. Such monitoring has enormous significance for eliminating the possibility of transmission of PERV infection, thus contributing to higher levels of safety in xenotransplantation.
Collapse
Affiliation(s)
- Joanna Gola
- Department of Molecular Biology, Medical University of Silesia, Jednosci 8, 41-200 Sosnowiec, Poland.
| | - Urszula Mazurek
- Department of Molecular Biology, Medical University of Silesia, Jednosci 8, 41-200 Sosnowiec, Poland.
| |
Collapse
|
|
9
|
Jung YD, Lee JR, Kim YJ, Ha HS, Oh KB, Im GS, Choi BH, Kim HS. Promoter activity analysis and methylation characterization of LTR elements of PERVs in NIH miniature pig. Genes Genet Syst 2014; 88:135-42. [PMID: 23832305 DOI: 10.1266/ggs.88.135] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
The potential risk of porcine endogenous retrovirus (PERV) transmission is an important issue in xenotransplantation (pig-to-human transplantation). Long terminal repeats (LTRs) in PERV elements show promoter activity that could affect neighboring functional genes. The methylation status and promoter activities of 3 LTR structures (PERV-LTR1, LTR2, and LTR3 elements) belonging to the PERV-A family were examined using luciferase reporter genes in human liver cell lines (HepG2 and Hep3B). The PERV LTR3 element exhibited hypomethylation and stronger promoter activity than the other LTR elements in human liver cells. We also performed comparative sequences analysis of the PERV LTR elements by using bioinformatics tools. Our findings showed that several transcription factors such as Nkx2-2 and Elk-1 positively influenced the high transcriptional activity of the PERV LTR3 element.
Collapse
Affiliation(s)
- Yi-Deun Jung
- Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea
| | | | | | | | | | | | | | | |
Collapse
|
|
10
|
Li ZG, Liu GB, Pan MX, Wu QS, Ge M, Du J, Wang Y, Gao Y. Knockdown of porcine endogenous retroviruses by RNA interference in Chinese experimental miniature pig fibroblasts. Transplant Proc 2013; 45:748-55. [PMID: 23498816 DOI: 10.1016/j.transproceed.2012.03.068] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2011] [Revised: 01/30/2012] [Accepted: 03/06/2012] [Indexed: 11/27/2022]
Abstract
BACKGROUND The clinical application of porcine-derived xenotransplants is limited by the potential risk of infection due to the presence of porcine endogenous retrovirus (PERV) in tissues, organs, and cells. The establishment of pig fibroblasts with low PERV expression and without PERV-C can provide a nuclear donor to generate a safer transgenic pig. METHODS In this study, we obtained Chinese Experimental Miniature Pig fibroblasts (CEMPF) with low expression of PERV and none of PERV-C. We designed small interfering RNA (siRNA) expressed as short hairpin RNAs (shRNA) based on the highly conserved gag and pol regions of PERV and screened for the most effective siRNA to inhibit PERV expression. The selected shRNA-pol3 fragment was introduced into the CEMPF to obtain an engineered CEMPF stably expressing shRNA-pol3. RESULTS The PERV mRNA expression level in the engineered CEMPF was only 7.9% of that observed in fibroblasts from wild-type CEMPF, PERV P15E protein expression was significantly reduced. HEK293 cells cocultured with the supernate of the engineered CEMPF showed no PERV infection. CONCLUSIONS Engineered CEMPF, which possess no risk of PERV-A/C infection, can serve as a nuclear donor to generate xenograft donor pigs.
Collapse
Affiliation(s)
- Z-G Li
- Department of Hepatobiliary Surgery, Zhujiang Hospital, Guangzhou, PR China
| | | | | | | | | | | | | | | |
Collapse
|
|
11
|
Scobie L, Padler-Karavani V, Le Bas-Bernardet S, Crossan C, Blaha J, Matouskova M, Hector RD, Cozzi E, Vanhove B, Charreau B, Blancho G, Bourdais L, Tallacchini M, Ribes JM, Yu H, Chen X, Kracikova J, Broz L, Hejnar J, Vesely P, Takeuchi Y, Varki A, Soulillou JP. Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts. THE JOURNAL OF IMMUNOLOGY 2013; 191:2907-15. [PMID: 23945141 DOI: 10.4049/jimmunol.1301195] [Citation(s) in RCA: 102] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Acellular materials of xenogenic origin are used worldwide as xenografts, and phase I trials of viable pig pancreatic islets are currently being performed. However, limited information is available on transmission of porcine endogenous retrovirus (PERV) after xenotransplantation and on the long-term immune response of recipients to xenoantigens. We analyzed the blood of burn patients who had received living pig-skin dressings for up to 8 wk for the presence of PERV as well as for the level and nature of their long term (maximum, 34 y) immune response against pig Ags. Although no evidence of PERV genomic material or anti-PERV Ab response was found, we observed a moderate increase in anti-αGal Abs and a high and sustained anti-non-αGal IgG response in those patients. Abs against the nonhuman sialic acid Neu5Gc constituted the anti-non-αGal response with the recognition pattern on a sialoglycan array differing from that of burn patients treated without pig skin. These data suggest that anti-Neu5Gc Abs represent a barrier for long-term acceptance of porcine xenografts. Because anti-Neu5Gc Abs can promote chronic inflammation, the long-term safety of living and acellular pig tissue implants in recipients warrants further evaluation.
Collapse
Affiliation(s)
- Linda Scobie
- Department of Life Sciences, Glasgow Caledonian University, Glasgow G4 0BA, United Kingdom
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
12
|
Lin X, Qi L, Li Z, Chi H, Lin W, Wang Y, Jiang Z, Pan M, Gao Y. Susceptibility of human liver cells to porcine endogenous retrovirus. EXP CLIN TRANSPLANT 2013; 11:541-5. [PMID: 23901808 DOI: 10.6002/ect.2012.0213] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
OBJECTIVES The risk of porcine endogenous retrovirus infection is a major barrier for pig-to-human xenotransplant. Porcine endogenous retrovirus, present in porcine cells, can infect many human and nonhuman primate cells in vitro, but there is no evidence available about in vitro infection of human liver cells. We investigated the susceptibility of different human liver cells to porcine endogenous retrovirus. MATERIALS AND METHODS The supernatant from a porcine kidney cell line was added to human liver cells, including a normal hepatocyte cell line (HL-7702 cells), primary hepatocytes (Phh cells), and a liver stellate cell line (Lx-2 cells), and to human embryonic kidney cells as a reference control. Expression of the porcine endogenous retrovirus antigen p15E in the human cells was evaluated with polymerase chain reaction, reverse transcription-polymerase chain reaction, and Western blot. RESULTS The porcine endogenous retrovirus antigen p15E was not expressed in any human liver cells (HL-7702, Phh, or Lx-2 cells) that had been exposed to supernatants from porcine kidney cell lines. Porcine endogenous retrovirus-specific fragments were amplified in human kidney cells. CONCLUSIONS Human liver cells tested were not susceptible to infection by porcine endogenous retrovirus. Therefore, not all human cells are susceptible to porcine endogenous retrovirus.
Collapse
Affiliation(s)
- Xinzi Lin
- Department of Hepatobiliary Surgery, Zhujiang Hospital, China
| | | | | | | | | | | | | | | | | |
Collapse
|
|
13
|
Xiang S, Ma Y, Yan Q, Lv M, Zhao X, Yin H, Zhang N, Jia J, Yu R, Zhang J. Construction and characterization of an infectious replication competent clone of porcine endogenous retrovirus from Chinese miniature pigs. Virol J 2013; 10:228. [PMID: 23837947 PMCID: PMC3718662 DOI: 10.1186/1743-422x-10-228] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2012] [Accepted: 05/30/2013] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Xenotransplantation from animals has been considered to be a preferable approach to alleviate the shortage of human allografts. Pigs are the most suitable candidate because of the anatomical and physiological similarities shared with humans as well as ethical concerns. However, it may be associated with the risk of transmission of infectious porcine pathogens. Porcine endogenous retroviruses (PERVs) are of particular concern because they have been shown to infect human cells in vitro. To date, researches on the molecular characteristics and potential pathogenicity of PERV are still tenuous. In this report, an infectious replication competent clone of PERV from Wuzhishan pigs (WZSPs) in China was generated and characterized. This infectious clone will contribute to studies on PERV virology and control of PERV in xenotransplantation using Chinese miniature pigs. METHODS The proviral DNA of PERV from WZSPs was amplified in two overlapping halves. Then the two fragments were isolated, subcloned and fused to generate pBluescriptαSK+-WZS-PERV recombinant clones. Screened with RT-PCR, a molecular clone of PERV designated as WZS-PERV(2) was selected. Its infectivity and replication competency were characterized in HEK293 cells by PCR, real-time fluorescent quantitative RT-PCR, western blot, indirect immunofluorescence assay as well as sequence analysis. RESULTS The ability of WZS-PERV(2) to infect human cells and produce infectious virions were shown after transfection of the clone into HEK293 cells and infection of PERV derived from this recombinant clone. The expression of Gag proteins were detected in HEK293 cells infected with the virus derived from the clone by the indirect immunofluorescence assay and western blot. The results of sequences analysis and comparison combined with the PCR based genotyping result demonstrated that the WZS-PERV(2) belonged to PERV-A subgroup. Compared with a previous proviral DNA clone of PERV (PERV-WZSP), G to A hypermutation occurred in the env gene of WZS-PERV(2) was found, whereas APOBEC proteins have the potential to inhibit the replication of a variety of retroviruses through a cDNA cytosine deamination mechanism, so we presumed these G to A hypermutation might be the contribution of porcine APOBEC3F. CONCLUSIONS Altogether, an infectious replication competent clone of PERV from Chinese miniature pigs (WZSPs) termed WZS-PERV(2) was generated, characterized and sequenced.
Collapse
Affiliation(s)
- Silong Xiang
- Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
14
|
Characterization of molecular clones of porcine endogenous retrovirus-A containing different numbers of U3 repeat boxes in the long terminal repeat region. J Virol Methods 2012; 181:103-8. [DOI: 10.1016/j.jviromet.2012.01.023] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2011] [Revised: 12/23/2011] [Accepted: 01/31/2012] [Indexed: 11/19/2022]
|
|
15
|
Multiplex high resolution melting assay for estimation of Porcine Endogenous Retrovirus (PERV) relative gene dosage in pigs and detection of PERV infection in xenograft recipients. J Virol Methods 2011; 175:95-100. [PMID: 21545811 DOI: 10.1016/j.jviromet.2011.04.026] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2010] [Revised: 04/19/2011] [Accepted: 04/20/2011] [Indexed: 11/20/2022]
Abstract
Porcine Endogenous Retrovirus (PERV) poses an infectious risk in the field of xenotransplantation. This risk may be mitigated by breeding selectively animals bearing favorable PERV genetic characteristics including pigs with low levels of PERV integrated in the genome. A real-time quantitative polymerase chain reaction (PCR) assay employing the Roche High Resolution Melting (HRM) Master was used to estimate the relative gene dosage of PERV pol integrated within the pig genome. When assessed across 99 pigs of the Auckland Island breed numerous animals bearing low gene dosage were identified. The assay was adapted further to perform multiplex PCR for the detection of PERV infection within xenograft recipients. Besides PERV, amplification targets for the multiplex PCR include a pig cell marker for the determination of microchimerism and an internal amplification control (IAC) to assess the efficiency of nucleic acid isolation and effects of PCR inhibition. When 12 patients who had received porcine islet transplants were tested no evidence of PERV infection was found. The assay was shown to be specific, highly reproducible with superior performance over conventional nested PCR. This assay can be used as both a screening tool for PERV proviral levels within donor pigs and as a diagnostic tool to examine PERV transmission in human patients treated with porcine xenotransplantation material.
Collapse
|
|
16
|
Current development of bioreactors for extracorporeal bioartificial liver (Review). Biointerphases 2011; 5:FA116-31. [PMID: 21171705 DOI: 10.1116/1.3521520] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
The research and development of extracorporeal bioartificial liver is gaining pace in recent years with the introduction of a myriad of optimally designed bioreactors with the ability to maintain long-term viability and liver-specific functions of hepatocytes. The design considerations for bioartificial liver are not trivial; it needs to consider factors such as the types of cell to be cultured in the bioreactor, the bioreactor configuration, the magnitude of fluid-induced shear stress, nutrients' supply, and wastes' removal, and other relevant issues before the bioreactor is ready for testing. This review discusses the exciting development of bioartificial liver devices, particularly the various types of cell used in current reactor designs, the state-of-the-art culturing and cryopreservation techniques, and the comparison among many today's bioreactor configurations. This review will also discuss in depth the importance of maintaining optimal mass transfer of nutrients and oxygen partial pressure in the bioreactor system. Finally, this review will discuss the commercially available bioreactors that are currently undergoing preclinical and clinical trials.
Collapse
|
|
17
|
The rapid production of high-titer porcine endogenous retrovirus(PERV)-B env pseudotype and construction of an EGFP-expressing replication competent PERV-A vector. J Virol Methods 2010; 171:61-6. [PMID: 20933542 DOI: 10.1016/j.jviromet.2010.09.030] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2010] [Revised: 09/29/2010] [Accepted: 09/30/2010] [Indexed: 11/22/2022]
Abstract
Porcine endogenous retroviruses (PERVs) present a unique concern associated with xenotransplantation because they have been shown to infect certain human cells in vitro and it is also difficult to generate herds of pigs free of PERVs. A simple system for the production of high-titer MoMLV-PERV pseudotypes is reported; an EGFP-expressing replication-competent molecular clone that allows direct measurement of titer was also constructed. To improve the MLV-based retroviral vector system, a 2.1-kb PERV-B env product was amplified from PK-15 genomic DNA and cloned into the pCL-Eco retroviral vector. The titer of lacZ (PERV-B) from the 293 cells was about 1.0×10(4) CFU/ml. In contrast, the titer of lacZ (PERV-B) from a conventional murine retroviral vector (split genome) was found to be 1.2×10(2) CFU/ml when the PERV-B env expression vector was transfected into TELCeB6 cells, which harbor MFGnlslacZ and the gag-pol-expressing vector. In addition, an infectious PERV-A clone containing enhanced GFP (EGFP) by using a PCR-based method was developed. This EGFP-expressing PERV-A-IRES-EGFP molecular clone was found to be stable genetically on transfection in 293 cells.
Collapse
|
|
18
|
Valdes-Gonzalez R, Dorantes LM, Bracho-Blanchet E, Rodríguez-Ventura A, White DJG. No evidence of porcine endogenous retrovirus in patients with type 1 diabetes after long-term porcine islet xenotransplantation. J Med Virol 2010; 82:331-4. [PMID: 20029803 DOI: 10.1002/jmv.21655] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Xenotransplantation is a promising alternative for donor shortage to ameliorate physiologic and metabolic disorders. The major concern for xenotransplant is the risk of zoonosis mainly by the porcine endogenous retrovirus (PERV), presentation in the piglet genome. Twenty-three patients with type 1 diabetes were transplanted with porcine islets using collagen-generating devices which were implanted subcutaneously in the anterior wall of the abdomen. Clinical characteristics and metabolic tests were recorded in each visit. They were tested for PERV using PCR and RT-PCR from blood pretransplantation and every 3 months during a 4.6- to 8-year follow-up after their first xenotransplant. Tests by PCR of every DNA sample (780 samples) revealed that there was no PERV infection in the DNA of white cells. No evidence of PERV activation was found in this group of patients with type 1 diabetes during clinical long-term follow-up.
Collapse
|
|
19
|
Frühauf JH, Mertsching H, Giri S, Frühauf NR, Bader A. Porcine endogenous retrovirus released by a bioartificial liver infects primary human cells. Liver Int 2009; 29:1553-61. [PMID: 19686312 DOI: 10.1111/j.1478-3231.2009.02087.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND Porcine endogenous retrovirus (PERV) remains a safety risk in pig-to-human xenotransplantation. There is no evidence of in vivo productive infection in humans because PERV is inactivated by human serum. However, PERV can infect human cell lines and human primary cells in vitro and inhibit human immune functions. AIMS We investigated the potential of primary porcine liver cells to transmit PERV to primary human cells in a bioreactor-based bioartificial liver (BAL). METHODS Primary human hepatocytes, endothelial cells and the human cell line HEK 293 were exposed to supernatants from BAL or from the porcine cell line PK-15. PERV polymerase-specific reverse-transcriptase polymerase chain reaction (RT-PCR) and PCR were used to investigate PERV transmission to human cells. An assay of RT activity was used to detect the presence of retrovirus in the supernatants of BAL, primary human hepatocytes and endothelial cells. RESULTS Primary human hepatocytes (hHep), endothelial cells and HEK 293 cells were reproducibly infected by PERV, originating from primary porcine liver cells within the BAL and from PK-15 cells. Infected cells were positive for PERV-specific DNA and RNA after 8-10 days on an average, and RT activity was detectable in the supernatants of infected hHep and HEK 293 cells. CONCLUSION A risk of PERV infection in human cells is documented in this study, indicating that short-term contact of primary porcine liver cell supernatants with primary human cells could result in PERV transmission.
Collapse
Affiliation(s)
- Jan-Henning Frühauf
- Department of Cell Techniques and Applied Stem Cell Biology, Biomedical-Biotechnological Center (BBZ), Leipzig, Germany
| | | | | | | | | |
Collapse
|
|
20
|
Specke V, Plesker R, Wood J, Coulibaly C, Suling K, Patience C, Kurth R, Schuurman HJ, Denner J. No in vivo infection of triple immunosuppressed non-human primates after inoculation with high titers of porcine endogenous retroviruses. Xenotransplantation 2009; 16:34-44. [PMID: 19243559 DOI: 10.1111/j.1399-3089.2009.00508.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
UNLABELLED Porcine endogenous retroviruses (PERVs) released from pig tissue can infect selected human cells in vitro and therefore represent a safety risk for xenotransplantation using pig cells, tissues, or organs. Although PERVs infect cells of numerous species in vitro, attempts to establish reliable animal models failed until now. Absence of PERV transmission has been shown in first experimental and clinical xenotransplantations; however, these trials suffered from the absence of long-term exposure (transplant survival) and profound immunosuppression. METHODS We conducted infectivity studies in rhesus monkeys, pig-tailed monkeys, and baboons under chronic immunosuppression with cyclosporine A, methylprednisolone, and the rapamycin derivative. These species were selected because they are close to the human species and PERVs can be transmitted in vitro to cells of these species. In addition, the animals received twice, a C1 esterase inhibitor to block complement activation before inoculation of PERV. In order to overcome the complications of microchimerism, animals were inoculated with high titers of cell-free PERV. In addition, to enable transmission via cell-cell contact, some animals also received virus-producing cells. For inoculation the primate cell-adapted strain PERV/5 degrees was used which is characterized by a high infectious titer. Produced on human cells, this virus does not express alpha 1,3 Gal epitopes, does not contain porcine antigens on the viral surface and is therefore less immunogenic in non-human primates compared with pig cell-derived virus. Finally, we present evidence that PERV/5 degrees productively infects cells from baboons and rhesus monkeys. RESULTS In a follow-up period of 11 months, no antibody production against PERV and no integration of proviral DNA in blood cells was observed. Furthermore, no PERV sequences were detected in the DNA of different organs taken after necropsy. CONCLUSION These results indicate that in a primate model, in the presence of chronic immunosuppression, neither the inoculation of cell-free nor cell-associated PERV using a virus already adapted to primate cells results in an infection; this is despite the fact that peripheral blood mononuclear cells of the same animals are infectible in vitro.
Collapse
|
|
21
|
Ramsoondar J, Vaught T, Ball S, Mendicino M, Monahan J, Jobst P, Vance A, Duncan J, Wells K, Ayares D. Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs. Xenotransplantation 2009; 16:164-80. [DOI: 10.1111/j.1399-3089.2009.00525.x] [Citation(s) in RCA: 113] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
|
|
22
|
Dieckhoff B, Kessler B, Jobst D, Kues W, Petersen B, Pfeifer A, Kurth R, Niemann H, Wolf E, Denner J. Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation. Xenotransplantation 2009; 16:64-73. [DOI: 10.1111/j.1399-3089.2009.00515.x] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
|
|
23
|
Thanos CG, Elliott RB. Encapsulated porcine islet transplantation: an evolving therapy for the treatment of type I diabetes. Expert Opin Biol Ther 2009; 9:29-44. [PMID: 19063691 DOI: 10.1517/14712590802630666] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
BACKGROUND Allogeneic tissue-based therapies for Type I diabetes have demonstrated efficacy but are limited due to tissue-sourcing constraints, as the number of patients exceeds that of tissue donors. Porcine islets derived from designated pathogen-free sources could be an alternative, particularly if delivered in a way that evades the host immune system's rejection. METHODS This review focuses on approaches designed to protect xenogeneic islets from immune rejection by provision of perm-selective barriers. RESULTS Designated pathogen-free herds could provide a supply of wild-type porcine islets that are well tolerated when administered in a suitable protective delivery vehicle. Such barrier systems have enabled amelioration of diabetes in a variety of animal models and preliminary evidence suggests that similar results could be attained in humans. CONCLUSION With advances in biomaterial design, source tissue selection, and the evolution of critical cell processing techniques, contemporary encapsulated porcine islet therapies offer a new level of clinical promise.
Collapse
Affiliation(s)
- C G Thanos
- Brown University, Department of Molecular Pharmacology, Physiology and Biotechnology, Providence, RI 02912, USA.
| | | |
Collapse
|