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The Lancet on Xenotransplantation - Proceed with Care
The Lancet on Xenotransplantation - Proceed with Care
The Lancet (29 August 1998) published three research articles
on xenotransplantation, two of which reported no infection in actual cases
of xenotransplantation, and one speculating on possible risks. The first
two articles were based on the complete absence of pig endogenous retrovirus
(PERV) infection in people who have received living pig tissue. The paper
that discusses the risks of xenotransplantation is based on speculation
and in-vitro observations, not on data showing in-vivo PERV infection in
any species. Although there is little in this latter paper that applies
to islet xenografts, it is important to note the main conclusions
drawn by the researchers:
| Our data are strong evidence for a viral risk but we do not know whether the titre will be high enough to bring about productive infection in vivo. Xenotransplant patients, however, will need long-term, intensive immunosuppression, increasing considerably the risk for viral xenozoonosis. Another disadvantage is that grafted organs will probably be obtained from pigs that are transgenic for human complement-regulators which may suppress complement-mediated virolysis,a natural protection against zoonosis. |
There are some key points here which do not apply to islet xenografts, and which must be highlighted when discussing any risks associated with xenotransplantation in general:
The arguments in this paper strengthen the case for islet xenografts being the best first candidate for xenotransplantation. If xenotransplantation proves successful in restoring normoglycemia to people with diabetes, the whole field will be advanced dramatically. Are you listening, Novartis?
Xenotransplantation Articles
in The Lancet - 29 August 1998
Xenotransplantation and Risk of Infection
No evidence of infection with porcine endogenous
retrovirus in recipients of porcine islet-cell xenografts
No evidence of pig DNA or retroviral infection
in patients with short-term extracorporeal connection to pig kidneys
Expression of pig endogenous retrovirus by primary
porcine endothelial cells and infection of human cells
No clear answers on safety of pigs as tissue
donor source
Other Xenotransplantation Links
The Case for Islet Xenografts being the
Best First Candidate for Xenotransplantation
The Xenotransplantation Debate - Science or Superstition?
Xenotransplantation is safe! CDC Report and Latest
Xeno Events
BBC News Online -- Pig
Viruses Don't Pass to Humans (August 8 1998)
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Volume 352, Number 9129 29 August 1998 |
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XENOTRANSPLANTATION AND RISK OF INFECTION |
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In this week's Lancet, three research groups report new findings on PERV. In the first report, Dr Ulrich Martin and colleagues from Germany report that PERV is also produced by cells from pig aortas, livers, lung, and skin-all tissues that are likely to be used for transplants. The findings, say the researchers, suggest "a serious risk of retrovirus transfer after xenotransplantaion". In the second study, however, Walid Heneine and colleagues from the USA and Sweden report that they found no evidence of PERV infection in blood samples from ten Swedish diabetes patients who had received transplants of insulin-producing cells from pigs-even though the patients had been exposed to large numbers of pig cells (400 million to 2 billion) and had been treated with drugs that should have reduced their ability to resist PERV infection (immunosuppressive drugs). In the final paper, Clive Patience and colleagues from the UK and Sweden write that they could find no evidence of PERV infection in two kidney-failure patients who had their blood passed through pig kidneys. In his Commentary (p 666), Jonathan Stoye from London, UK, argues that only with limited clinical trials, which regulatory authorities are now moving towards permitting, "will it be possible to test long-term xenograft survival and function".
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No evidence of infection with porcine endogenous retrovirus in recipients of porcine islet-cell xenografts |
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Summary
Introduction
Patients
and Methods
Results
Discussion
References
Methods We studied 10 diabetic patients who had received porcine fetal islets between 1990 and 1993, looking for evidence of PERV infection by using PCR serology, PCR, and reverse transcriptase assays. Prolonged xenograft survival (up to a year) was confirmed in five patients by porcine C-peptide excretion and detection of pig mitochondrial DNA (mtDNA) in serum.
Findings Despite the evidence for extended exposure to pig cells and despite concomitant immunosuppressive therapy, we were unable to detect markers of PERV infection in any patient. Screening for two PERV sequences in peripheral blood lymphocytes collected 4-7 years after the xenotransplantation was negative. Markers of PERV expression, including viral RNA and reverse transcriptase, were undetectable in sera from both early (day 3 to day 180) and late (4-7 years) time points. Western blot analysis for antibodies was consistently negative.
Interpretation These results suggested the absence of PERV infection in these patients. Also this study establishes a minimum standard for post-transplant surveillance of patients given porcine xenografts.
Lancet 1998; 352: 695-99
Retroviruses result in lifelong infection12 and reports that PERV from cell lines and porcine lymphocytes can infect human cells in vitro8,10 have prompted the US Food and Drug Administration to put porcine xenograft trials on hold until previously exposed patients are assessed for PERV infection and until prospective monitoring of xenograft recipients is established.
We have studied 10 Swedish patients, transplanted with porcine fetal pancreatic islets between 1990 and 1993, for evidence of infection with PERV. These patients have been exposed to a large number of pig cells and xenograft survival has been prolonged. All these patients had a rise in antipig antibody titre within a month of transplantation.13,14
Lysate containing DNA from 150 000 PBLs was subjected to 35 cycles of amplification followed by Southern blot hybridisation to 32P end-labelled internal oligoprobes PK15GP1 and PRETP2 for the pol and gag sequences, respectively. Negative controls included water and uninfected human PBL lysate. Pig PK15 cell lysates were 10-fold serially diluted with uninfected human PBL lysate to 1·5 and 0·15 PK15 cells as positive controls. The PCR reaction with the DNA equivalent of 0·15 PK15 cell represents the detection limit of the assay.
*Sequences obtainable from WH.
| Patient | Pig mtDNA* | Transplant characteristics |
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| 2-3 days | 2 wk | 3 wk | 6 mo | 1 yr | 4-7 yr |
Evidence of | Site | ICCs (1000s) | |
| xenoislet survival | |||||||||
| XIT1 | + | + | + | + | - | - | C-peptide+ | IP | 390 |
| XIT2 | + | - | - | - | NA | NA | IP | 520 | |
| XIT3 | - | NA | - | - | NA | - | IP | 460 | |
| XIT4 | + | - | - | - | NA | - | IP | 410 | |
| XIT5 | + | - | - | - | NA | - | IP | 330 | |
| XIT6 | - | - | - | - | NA | - | C-peptide+ | IP | 520 |
| XIT7 | + | - | - | + | + | - | C-peptide+ | IP | 800 |
| XIT8 | + | + | - | + | - | - | C-peptide+ | IP | 1020 |
| XIT9 | - | - | - | - | NA | - | RC | 200 | |
| XIT10 | - | + | - | + | NA | NA | Biopsy+ | RC | 410 |
| *PCR results at time after xenotransplantation; NA=samples not available. | |||||||||
| IP=intraportal;
RC=renal capsule; ICC=islet-like cell clusters; C-peptide+=urinary excretion
of porcine C-peptide detected; biopsy+=detection of pig cells under renal
capsule in biopsy 3 weeks post-tranplant. |
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| 4-7
year results are for samples collected in both April and August, 1997. |
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| Table 1: PCR analysis of pig mtDNA sequences in sera from 10 diabetic patients given pig islet cells | |||||||||
PBLs collected from patients XIT2 and XIT10 at one time point and from
the other eight patients at two or three time points 32-86 months post-transplantation
were all negative for both gag and pol PERV sequences (table
2, figure 1).
| Post-transplantation | Time of sampling | PERV sequences* | |
| gag | pol | ||
| 32-60 mo | April, 1995 | 0/9 | 0/9 |
| 32-60 mo | April, 1997 | 0/8 | 0/8 |
| 32-60 mo | April, 1997 | 0/8 | 0/8 |
| *Number of positive samples/total tested; sample from XIT6 not available in April, 1995 and XIT2 and XIT10 not available in April and August, 1997 | |||
| Table 2: PCR analysis of PERV proviral sequences in lymphocytes from 10 diabetic patients given pig islet-cell xenografts between June, 1990 and April, 1993 | |||
Sera were also tested for porcine mtDNA, a pig-specific cellular marker
that is more sensitive than single-copy genomic sequences. Pig mtDNA was
detectable from day 3 in six patients and up to one year in patient XIT7
(table 1). Among patients tested 4-7 years after the xenotranplant, none
had detectable pig mtDNA (figure 2). The pig mtDNA PCR signals were strongest
at day 3, consistent with higher levels of pig-source cells or cellular
products in patients' blood during this early period. The prevalence of
detectable pig mtDNA in sera was also highest at 3 days in six of 10 patients
and decreased gradually. However, the presence of pig mtDNA in sera of
four patients 6 months after transplant argues for successful persistence
of pig cells. The inconsistent detection at intermediate time points in
three patients probably reflects the low fluctuating levels of porcine
mitochondria in the small volumes (25 µL) of sera tested. Transplant
technique may also have an influence: porcine mtDNA was detectable at 3
days in six of eight patients who received intraportal transplants but
in neither of the two with pig cells implanted under the renal capsule.
Monitoring urinary excretion of porcine C-peptide, as a marker of xenograft
persistence, was generally concordant with the mtDNA findings. Urinary
porcine C-petide was detected for more than 6 months but not in any of
the five who were persistently porcine mtDNA-negative after day 3.
Antibodies to p30 PERV protein were not detected in any serum collected
around 6 months post-transplantation from 10 patients. Additional samples
collected from eight patients from two time points 4-7 years after transplantation
were also seronegative. Sera from two pigs also tested negative, confirming
immunological tolerance to PERV proteins, as expected with an endogenous
virus (figure 3).
The potential for exposure to PERV from the xenograft may be highest during the first 6 months. Viraemia after exposure to other retroviruses (eg, HIV-1) is commonly seen during this time.24-26 However, we found no evidence of PERV RNA in patients' sera during this period, arguing against transient or abortive primary productive PERV infections.
Detection of PERV DNA transcripts in cellular RNA from pig tissues does not show that such expression yields detectable cell-free virus production in serum.8,9 Our findings of both PERV RNA and RT activity in sera from 75% of tested pigs demonstrate productive release of PERV virions into the serum, and imply that any PERV infection in man could also be associated with detectable cell-free virus in serum. These findings highlight the diagnostic importance of using serum PERV RNA and RT levels as markers of PERV expression.
The rapid clearance of porcine cells from five patients may explain the failure of PERV to establish infection. However, excretion of porcine C-peptide by four patients up to 450 days after the transplantation combined with the finding of porcine mitochondrial sequences in serum for 6 months to one year in four patients argue for extended graft survival in at least five patients. Another factor that might influence the potential for PERV to transmit from xenograft to man is a generally low PERV infectivity for human cells, and little or no PERV expression by porcine ICCs.8 We have started to analyse the kinetics of PERV expression in ICCs and preliminary results confirm both PERV RNA and RT activity in the supernatants of cultured fetal pig ICCs after 2 and 4 days in culture. However, the ability of this ICC-derived PERV to infect human cells has not been studied yet. Host factors, including complement-mediated viral lysis, may also protect against PERV infection. Pig-cell-derived PERV are inactivated and lysed by human sera.8 The xenografts were not procured from transgenic pigs carrying human complement-inhibition factors2 and no attempt was made to remove preformed xenoantibodies13,14 or to block complement activation. Complement-based inactivation of PERV released from the pig xenograft should not, therefore, have been compromised.
The absence of detectable RT is consistent with the absence of PERV RNA and, since RT is a generic marker for retroviruses, failure to detect it in these patients' sera points to the absence of any other, unrecognised, retrovirus of porcine origin. Our data cannot definitively exclude infection with retroviruses; nevertheless, the observations are reassuring. The risk that any xenograft recipient will become infected with PERV is likely to be a function of several factors associated with the source animal, xenotransplant technique, and the recipient's characteristics, so defining the risk will be complex. Nor can the results of any individual study be generalised to other types of exposure. However, this is an important initial evaluation of the risk for PERV infection after exposure to cellular xenografts from non-transgenic pigs. Furthermore, this study sets a standard for post-transplantation laboratory surveillance of PERV infection.
2 Groth CG, Korsgren O, Tibell A, J, et al. Transplantation of porcine fetal pancreas to diabetic patients. Lancet 1994; 344: 1402-04.
3 Deacon T, Schumacher J, Dinsmore J, et al. Histological evidence of fetal pig neural cell survival after transplantation into a patient with Parkinson's disease. Nat Med 1997; 3: 350-53.
4 Chari RS, Collins BH, Magee JC, et al. Treatment of hepatic failure with ex vivo pig-liver perfusion followed by liver transplantation. N Engl J Med 1994; 331: 234-37.
5 Chapman LE, Folks TM, Salomon DR, Patterson AP, Eggerman TE, Noguichi PD. Xenotransplantation and xenogenic infections. N Engl J Med 1995; 333: 1498-501.
6 Lieber MM, Sherr CJ, Benvensiste RE, Todaro GJ. Biologic and immunologic properties of porcine type C viruses. Virology 1975; 66: 616-19.
7 Suzuka I, Shimizu N, Sekiguchi K, Hoshino H, Kodama M, Shimotohno K. Molecular cloning of unintegrated closed circular DNA of porcine retrovirus. FEBA 1986; 198: 339-43.
8 Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an endogenous retrovirus of pigs. Nat Med 1997; 3: 282-86.
9 Le Tissier P, Stoye JP, Yasuhiro Y, Patience C, Weiss RA. Two sets of human-tropic pig retrovirus. Nature 1997; 389: 681-82.
10 Wilson CA, Wong S, Muller J, Davidson CE, Rose TM, Burd P. Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. J Virol 1998; 72: 3082-87.
11 Akiyoshi DE, Denaro M, Zhu H, Greenstein JL, Banerjee P, Fishman JA. Identification of a full length cDNA for an endogenous retrovirus of miniature swine. J Virol 1998; 72: 4503-07.
12 Coffin JM. Retroviridae and their replication. In: Fields BN, Knipe DM, Chanock RM, et al, eds. Fields virology, 2nd edn. New York: Raven, 1990: 1437-500.
13 Satake M, et al. Kinetics and character of xenoantibody formation in diabetic patients transplanted with fetal porcine islet cell clusters. Xenotranplantation 1994; 1: 24.
14 Galili U, Tibell A, Samuelsson B, Rydberg L, Groth CG. Increased anti-gal activity in diabetic patients transplanted with fetal porcine islet cell clusters. Transplantation 1995; 59: 1549-56.
15 Bjöersdorff A, Korsgen O, Feinstein R, et al. Microbiological characterization of porcine fetal islet-like cell clusters for intended clinical xenografting. Xenotransplantation 1995; 2: 26-31.
16 Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA. Analysis of enzymatically amplified B-globin and HLA-DQa DNA with allele-specific oligonucleotide probes. Nature 1986; 243: 163-66.
17 Mulder J, McKinney N, Christopherson C, Sninsky J, Greenfield L, Kwok S. Rapid and simple PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma: application to acute retroviral infection. J Clin Microbiol 1994; 32: 292-300.
18 Heneine W, Yamamoto S, Switzer WM, Folks TM. Detection of reverse transcriptase by a highly sensitive assay in sera from individuals infected with the human immunodeficiency virus type 1. J Infect Dis 1995; 171: 1210-16.
19 Garcia Lerma JG, Yamamoto S, Gomez-Cano M, et al. Measurement of human immunodeficiency virus type 1 plasma virus load based on reverse transcriptase (RT). J Infect Dis 1998; 177: 1221-29.
20 Yamamoto S, Folks TM, Heneine W. Highly sensitive qualitative and quantitative detection of reverse transcriptase activity. J Virol Methods 1996; 61: 135-43.
21 Sherr CJ, Fedele LA, Benventiste RE, Todaro GJ. Interspecies antigenic determinants of the reverse transcriptases and p30 proteins of mammalian type C viruses. J Virol 1975; 15: 1440-48.
22 Tibell A, Reinholt FP, Korsgren O, et al. Morphological identification of porcine islet cells three weeks after tranplantation to a diabetic patient. Transplant Proc 1994; 26: 1121.
23 Kaplan JE, Khabbaz RF. The epidemiology of human T lymphotropic virus types I and II. Rev Med Virol 1993; 3: 137-48.
24 Busch MP, Satten GA. Time course of viremia between exposure and seroconversion in health care workers with occupationally acquired infection with human immunodeficiency virus. Am J Med 1997; 102: 117-24.
25 Watson A, Ranchalis J, Travis B, et al. Plasma viremia in macaques infected with simian immunodeficiency virus. J Virol 1997; 71: 284-90.
26 Lutz H, Pedersen NC, Theilen GH. Course of feline leukaemia virus infection and its detection by enzyme-linked immunosorbent assay and monoclonal antibodies. Am J Vet Res 1983; 44: 2054-59.
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No evidence of pig DNA or retroviral infection in patients with short-term extracorporeal connection to pig kidneys |
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Summary
Introduction
Patients
and methods
Results
Discussion
References
Methods We took serial blood samples from two renal dialysis patients whose circulation had been linked extracorporeally to pig kidneys and tested them for pig DNA and PERV DNA by nested PCR. The patients' plasma was also tested for neutralising antibodies to two anthropotropic PERV strains.
Findings Having established that the nested PCRs could detect single molecules of target sequence, we analysed DNA isolated from patients' peripheral blood mononuclear cells. We found no evidence of pig or PERV DNA in either patient, even in samples taken as early as 6 h after the perfusion. Furthermore, we found no evidence of seroconversion for PERV-specific antibodies.
Interpretation The absence of porcine cells in the circulation of both patients, even in the samples taken soon after the perfusion experiment, suggests that any porcine cells dislodged from the kidney became rapidly sequestered from the circulation. Since cell-to-cell contact increases the efficency of infection of PERV this removal of porcine cells may increase the risk of transmission of PERV to the xenograft recipient. We did not, however, detect indications of infection by PERV by PCR or neutralisation assay. The genetic and serological methods described here will be useful for detection of possible PERV infection in other patients.
Lancet 1998; 352: 699-701
We report sensitive techniques to detect porcine DNA and PERV genomes in two dialysis patients who had short-term extracorporeal vascular connection to pig kidneys.3,4
*Sequences obtainable from CP.
Three porcine sequences were targeted for PCR amplification: the mitochondrial cytochrome oxidase subunit II (COII) gene, the ß-globin gene, and the protease gene of PERV. We also report on an assay for neutralising antibodies to two distinct amphotropic PERV isolates5 in the patients' sera.
PCR primers* were designed to be specific for porcine sequences. Thermal cycling conditions were: 92°C for 4 min, 1 cycle; 94°C for 1 min, 55°C for 1 min 10 s, 72°C for 1 min, 30 cycles; and 72°C for 5 min, 1 cycle (Robocycler, Stratagene). First round (outer) reactions were set up in 50 µL volumes with 1 µL being transferred into the second round (inner) reactions. All DNA samples were tested in triplicate.
To search for neutralising antibodies to PERV envelope proteins, murine
leukaemia virus (MLV) vectors bearing PERV env were used. Open-reading
frames for PERV-A and PERV-B5 were individually cloned and transfected
into TELCeB6 cells, which produce a MLV core containing a LacZ vector genome.6
The human TELCeB6 packaging cell line ensured that Gal
1-3Gal
epitopes, which might have contributed to particle neutralisation, were
absent from the vector. Vector particles were harvested in serum-free Opti-MEM
and incubated with fresh or heat-inactivated samples of patient's plasma
in a 1:1 mixture at 37°C for 1 h. The virus/plasma mixture was then
titrated on mink Mv-1-Lu cells.
All DNA samples and PCR reagents were kept separated and in laboratories
where porcine DNA and cells had not been handled. Water control amplifications
were done in all assays and were always negative. We also differentiated
between amplicons arising from genomic sequences and positive control DNAs
by sequencing cloned PCR products of the three genes and linearising them
with restriction enzymes within the region amplified by the inner primer
pair. Nucleotides were then removed from the plasmid ends by nuclease digestion
and the deleted plasmids were religated and transformed into bacteria to
yield PCR products smaller than those from genomc DNA (figure, upper part).
To determine the number of porcine cells needed in the patient cell preparation to produce a positive result, porcine PBMC were titrated in 2×106 human PBMC, and 105 cell equivalents were analysed by PCR after DNA extraction. The PCR for COII could detect a single porcine cell; the PCRs for ß-globin and PERV required about 20 and 2 cells, respectively (figure, lower part). The results from these controls are similar in sensitivity to those of Heneine and Switzer.2
DNA was obtained from PBMC samples taken 6 h up to 36 months after the extracorporeal perfusion.3 DNA was prepared from 2×106 PHA-stimulated PBMC which had been cultured for up to 4 days. The DNA of 105 cell equivalents was analysed by PCR. Porcine DNA was not detected in the circulation of either patient by any primer pair, even in samples taken only 6 h after dialysis (table). Neither patient's plasma from the perfusion experiment nor control human plasma affected the titre of the PERV A or B vector particles, indicating the absence of neutralising antibodies to these PERV envelopes.
| Time post | PCR for | Anti-PERV | ||
| dialysis | PERV | COII | ß-globin | antibodies* |
| Pre-dialysis | . . | . . | . . | -- |
| 6 h | . . | . . | . . | NT |
| 7 days | . . | . . | . . | NT |
| 21/24 mo | . . | . . | . . | NT |
| 33/36 mo | . . | . . | . . | -- |
| In all cases positive controls indicated that PCR assays could detect near single copy number of target sequence. . .=negative PCR. | ||||
| *"--"titre after plasma treatment was the range of 70-130% of the titre after treatment with control plasma of culture medium for both PERV-A and PERV-B vectors; NT=not tested. | ||||
| Analysis of patient PBMC DNAs for porcine sequences and patient plasma for anti-PERV antibodies | ||||
Our nested PCR systems, which are sensitive enough to pick up single molecules of target porcine sequences, did not detect such sequences in the circulation of two patients who had been temporarily connected to porcine kidneys. Nor did we find evidence of PERV infection either by PCR (although in vitro PBMC are not readily infected1) or by seroconversion. Negative findings on just two patients must be interpreted with caution and, if xenotransplantation is to proceed, it will be important to monitor other exposed individuals. The methods described here will be useful for that purpose.
2 Heneine W, Switzer WM. Highly sensitive and specific polymerase chain reaction assays for detection of baboon and pig cells following xenotransplantation in humans. Transplantation 1996; 62: 1360-62.
3 Breimer ME, Björck E, Svalander CT, et al. Extracorporeal ("ex vivo") connection of pig kidneys to humans I: clinical data and studies of platelet destruction. Xenotransplantation 1996; 3: 328-39.
4 Rydberg L, Björck S, Hallberg E, et al. Extracorporeal ("ex vivo") connection of pig kidneys to humans II: the anti-pig antibody response. Xenotransplantation 1996; 3: 340-53.
5 Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss RA. Two sets of human-tropic pig retrovirus. Nature 1997; 389: 681-82.
6 Takeuchi Y, Patience C, Magre S, Banerges PT, le Tissier P, Stoye JP, Weiss RA. Host-range and interference studies on three classes of pig endogenous retrovirus. J Virol (in press).
7 Hoopes CW, Platt JL. A molecular epidemiological probe for pig microchimerism. Transplantation 1997; 64: 347-50.
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Expression of pig endogenous retrovirus by primary porcine endothelial cells and infection of human cells |
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Summary
Introduction
Materials
and methods
Results
Discussion
References
Methods We have analysed pig primary aortic endothelial cells (PAEC) together with other transplantation-relevant porcine cells and tissues for expression of PERV mRNA. Release of virus particles by PAEC was monitored by reverse transcriptase (RT) activity in the medium of cultured PAEC. Infectivity for human cells was tested by co-cultivation of irradiated PAEC with the human embryonal kidney cell line HEK293 and looking for virus release from the human cells.
Findings PAECs, hepatocytes, lung, and skin from a variety of pig strains and breeds expressed PERV mRNA. PAEC released infectious particles. Co-cultivation of PAEC and HEK293 led to productive infection of the human cells and expression of PERV types A and B.
Interpretation Release of infectious virus from PAEC occurred without mitogenic stimulation, suggesting a serious risk of retrovirus transfer after xenotransplantation.
Lancet 1998; 352: 692-94
*Sequences obtainable from UM.
An internal BMV-specific oligonucleotide* was labelled (DIG-oligonucleotide tailing kit, Boehringer Mannheim). The blot was hybridised in 5×SSC, 0·1% N-laurylsarcosine, 0·02% SDS, 1% blocking reagent (Boehringer Mannheim), 0·1 mg/mL Poly(A) (Boehringer Mannheim) at 50°C overnight with the digoxigenin (DIG)-tailed oligonucleotide, washed twice for 5 min at room temperature and twice for 15 min at 62°C in 2×SSC. Chemoluminescence was detected with the DIG luminescence kit (Boehringer Mannheim).
.gif) |
Human endothelial cells (HuEC) as negative control. One representative
RT-PCR result shown for six landbreed pigs from Germany.
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-galactosyltransferase
specific primers10 (not shown) PERV pol-specific PCR
could detect one PK15 cell in 105 HEK 293 cells; with porcine
ß-globin and -galactosyltransferase
specific primers, one PK15 cell could be detected in 104 HEK293
cells (not shown). PERV-specific sequences could also be detected with
newly designed PERV LTR/leader primers with 5-10 times lower sensitivity
than the porcine ß-globin specific primers (not shown).
We also examined PERV RNA expression in cocultured HEK293 cells by RT-PCR
and detected PERV type A and B mRNA (figure 4). Internal controls without
RT excluded contamination with pig genomic DNA. Sequence analysis of the
pol
cDNA fragment yielded a sequence identical to the published
pol
sequence.1 Strong RT activity in the supernatant of the cultured
cells (figure 4) confirmed productive infection of HEK293 cells.
In our cocultivation experiments, contamination with pig cells, still
viable despite the X-irradiation, would have caused false-positive PERV
results but we excluded this by control PCRs with pig-specific ß-globin
and 
-galactosyltransferase
primers. Quantitative comparison of the PERV amplification products with
the results of endpoint dilution experiments (PERV pol/LTR-leader
vs
porcine
D-galactosyltransferase and ß-globin specific PCR) ruled out residual
contamination by porcine DNA or persistent PAEC in the human cells.
Our results confirm recent reports of the activation of infectious endogenous pig virus and extend them to pig endothelial cells, a constitutive part of all vascularised xenografts. The high number of PERV gene copies (about 10-40) in the porcine genome2,3 will make the task of producing PERV-free swine difficult or impossible. Our data are strong evidence for a viral risk but we do not know whether the titre will be high enough to bring about productive infection in vivo. Xenotransplant patients, however, will need long-term, intensive immunosuppression, increasing considerably the risk for viral xenozoonosis. Another disadvantage is that grafted organs will probably be obtained from pigs that are transgenic for human complement-regulators which may suppress complement-mediated virolysis,12 a natural protection against zoonosis. If transmission of PERV does occur clinically, two outcomes are possible--either the retrovirus is pathogenic but is restricted to the recipient or the virus (pathogenic or not) could infect people not participating in the xenotransplantation process. Recombination with human retroviral elements might enhance the risk. Further studies, in suitable primate models, are thus necessary to assess the risk of PERV infection in vivo after xenotransplantation.
2 Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss RA. Two sets of human-tropic pig retrovirus. Nature 1997; 389: 681-82.
3 Wilson CA, Wong S, Muller J, Davidson CE, Rose TM, Burd P. Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. J Virol 1998; 72: 3082-87.
4 Bach FH, Fineberg HV. Call for moratorium on xenotransplants. Nature 1998; 391: 326.
5 Vogel G. No moratorium on clinical trials. Science 1998; 279: 648.
6 Weiss RA. Transgenic pigs and virus adaptation. Nature 1998; 391: 327-28.
7 Editorial. Halt the xeno-bandwagon. Nature 1998; 391: 309.
8 Editorial. Does biomedical research need another moratorium? Nat Med 1998; 4: 131.
9 Heneine W, Switzer HM. Highly sensitive and specific polymerase chain reaction assays for detection of baboon and pig cells following xenotransplantation in humans. Transplantation 1996; 62: 1360-62.
10 Hoopes CW, Platt JL. A molecular epidemiological probe for pig microchimerism. Transplantation 1997; 64: 347-50.
11 Silver J, Maudru T, Fujita K, Repaske R. An RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions Nucleic Acids Res 1993; 21: 3593-594.
12 Saifuddin M, Parker CJ, Peeples ME, et al. Role of virion-associated glycosylphosphatidylinositol-linked proteins CD55 and CD59 in complement resistance of cell line-derived and primary isolates of HIV-1. J Exp Med 1995; 182: 501-09.
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No clear answers on safety of pigs as tissue donor source |
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Experience with allotransplantation has provided ample evidence that donor organs can be a source of infection.3 This point has focused attention on the microbiological status of potential source animals, which is one reason for favouring pigs over primates for this role.4 However, even the advanced techniques developed to breed high-health-status pigs will not eliminate pathogens such as those capable of infecting fetuses congenitally or transmitted in the germline. Although congenitally acquired pathogens might prove important, it is the latter group of agents, known as the endogenous retroviruses, that are believed to pose the greatest threat of infectious disease to xenotransplantation. These viruses consist of retroviral elements that are inherited in a mendelian fashion after infection of germ cells during evolution.5 All vetebrates contain thousands of these elements. Most of the elements, apparently including all those present in human beings, seem defective, but some, including proviruses present in mice, cats, and chickens, will give rise to infectious retroviruses. These viruses can, but need not, cause "spontaneous" neoplasms in their host species.
Extrapolating from these observations, several researchers have suggested
that porcine endogenous retroviruses (PERVs) might be an obstacle in xenotransplantation.6-8
To constitute a serious threat there would probably have to be a chain
of events (panel). There is now substantial evidence for the first three
events in this chain.9-12 Two sets of pig retrovirus, PERV-A
and PERV-B, capable of in-vitro replication in certain human cell lines,
have been described. They are widely distributed in different pig breeds
and expressed in different tissues, including spleen, kidney, and heart.
The
paper by Ulrich Martin and colleagues adds further weight to these
conclusions. These investigators add four more cell or tissue types (aortic
endothelial cells, hepatocytes, skin, and lung) to the list of PERV mRNA
expressors and also show that primary aortic endothelial cells from several
different pig breeds produce infectious PERV-A and PERV-B. Briefly described
in the paper by Walid
Heneine and colleagues are preliminary data indicating that fetal porcine
islet cells also express PERVs. Because of the way these elements are inherited
in pigs, they will be virtually impossible to eliminate from source herds.
Taken together, these data show that most if not all transplanted porcine
tissues will express one or more retroviruses capable of infecting human
cells.
Does the presence of potentially infective retroviruses in procine tissue mean that infection of human beings exposed to porcine cells will inevitably follow? Several groups are investigating this question; the papers by Heneine and Clive Patience and their colleagues represent the first published reports of such studies. Both groups have developed sensitive methods for detecting PERV infection in vivo by use of PCR and assays for seroconversion. Heneine and colleagues examined ten diabetic patients who received porcine fetal islets. In no case was there any evidence for PERV infection, even though the xenograft survived a long time in five of the patients. Patience and colleagues examined two renal-dialysis patients with short-term extracorporeal vascular connnection to pig kidneys. Neither showed any sign of viral infection. Since rates of infection are likely to depend on a variety of factors--including the pig source, whether or not the pig carries transgenes designed to regulate complement activation,13 the nature of the xenograft, and the degree of immunosuppression--care must be taken not to draw too broad a set of conclusions from these two studies. Nevertheless, it seems reasonable to conclude that the PERVs will not show the very high levels of transmission associated with some viruses. Some unpublished studies involving other examples of exposure to pig tissues seem to be reaching the same conclusion. These studies ought to be made available for public scrutiny in the near future.
So far no conclusions can be drawn about the potential pathogenicity of PERVs if cross-species infection occurs. It has been suggested that trials in non-human primates might shed some light on this question; unfortunately recent results indicate that PERV-A and PERV-B will not recognise receptors on cells from non-human primates thereby providing evidence against the value of such studies.14
What is the significance of the findings reported today for the future of xenotransplantation? Some people will undoubtedly be concerned by the results of Martin and colleagues. They will argue that, so long as porcine xenografts express retroviruses capable of infecting human cells, clinical trials should not be allowed to proceed because any risk posed by these elements is unacceptably large in the absence of information about the pathogenic potential of the PERVs. Others will be reassured by the results of Heneine and Patience. They will argue that any such risks are worth taking, given the promise of xenotransplantation and the apparent low levels of infectivity of the PERVs.
The regulatory climate is moving toward permitting limited clinical trials in the near future, especially in the USA. Only with such trials will it be possible to test long-term xenograft survival and function. However, xenotransplantation is a term covering a wide range of different procedures; precautions that may be appropriate in one case may not be sufficient in another, and consequences that would be perfectly acceptable for individual patients would be unthinkable if they were to affect the general population. The clinical trials must therefore be conducted in a deliberate, stepwise fashion, using the recently developed assay methods to monitor PERV infection and to assess whether transmission to contacts can occur. These trials must involve close liaison between surgeons, immunologists, and virologists, and include lifetime monitoring by regulatory authorities.
Jonathan Stoye
1 Advisory Group on the Ethics of Transplantation. Animal tissues into humans. Norwich, UK: Stationery Office, 1997: 1-258.
2 Bach FH, Fishman JA, Daniels N, et al. Uncertainty in xenotransplantation: individual benefit versus collective risk. Nat Med 1998; 4: 141-44.
3 Michaels MG, Simmons RL. Xenotransplant-associated zoonoses. Transplantation 1994; 57: 1-7.
4 Fishman JA. Miniature swine as organ donors for man: strategies for prevention of xenotransplant-associated infections. Xenotransplantation 1994; 1: 47-57.
5 Boeke JD, Stoye JP. Retrotransposons, endogenous retroviruses, and the evolution of retroelements. In: Coffin JM, Hughes SH, Varmus HE, eds. Retroviruses. Cold Spring Harbor: Cold Spring Harbor Press, 1997: 343-435.
6 Smith DM. Endogenous retroviruses in xenografts. N Engl J Med 1993; 328: 142-43.
7 Stoye JP, Coffin JM. The dangers of xenotransplantation. Nat Med 1995; 1: 1100.
8 Chapman LE, Folks TM, Salomon DR, Patterson AP, Eggerman TE, Noguchi PD. Xenotransplantation and xenogeneic infections. N Engl J Med 1995; 333: 1498-501.
9 Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an endogenous retrovirus of pigs. Nat Med 1997; 3: 282-86.
10 Wilson CA, Wong S, Muller J, Davidson CE, Rose TM, Burd P. Type C retrovirus released from porcine primary peripheral blood mononuclear cells infects human cells. J Virol 1998; 72: 3082-87.
11 Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss RA. Two sets of human-tropic pig retrovirus. Nature 1997; 389: 681-82.
12 Akiyoshi DE, Denaro M, Zhu H, Greenstein JL, Banerjee P, Fishman JA. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J Virol 1998; 72: 4503-07.
13 Weiss RA. Transgenic pigs and virus adaptation. Nature 1998; 391: 327-28.
14 Takeuchi Y, Patience C, Magre et al. Host-range and interferon studies on three classes of pig endogenous retrovirus. J Virol (in press).
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plasmid controls. Amplifications from DNA of 50 cell equivalents or 50
copies of plasmid DNA. P=PERV protease; C=cytochrome oxidase; ß=ß-globin;
M=marker (sizes in base-pairs). All water controls negative.
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