Monitoring for Presence of Potentially Xenotic Viruses in Recipients of Pig Islet Xenotransplantation

Abstract

This study represents a long-term follow-up of human patients receiving pig islet xenotransplantation. Eighteen patients had been monitored for up to 9 years for potentially xenotic pig viruses: pig endogenous retrovirus, pig cytomegalovirus, pig lymphotropic herpesvirus, and pig circovirus type 2. No evidence of viral infection was found.

Human xenotransplant recipients are theoretically at risk of infection with animal viruses. Pig endogenous retroviruses (PERVs) have been a major source of anxiety, since they are present in the genome of all pigs and infect human cells (25, 29). Whether PERV truly poses an infection risk will ultimately be determined by in vivo monitoring of patients and experimental animals receiving xenografts. To date, more than 200 patients exposed to pig cells have been tested for evidence of PERV infection (3, 5, 14, 15, 16, 17, 21, 24, 26, 27, 33, 34). Additionally, some 50 nonhuman primates have also been tested for PERV infection (20, 32). No evidence of infection has been found. Three different species of nonhuman primates, which were heavily immunosuppressed and injected with a human cell-adapted virus, also failed to show any evidence of PERV transmission (4, 30). Similar results were obtained when PERVs were injected into small animals, regardless of whether they were immunosuppressed (29-31).

Other pig viruses are able to establish persistent infections or, alternatively, are potentially oncogenic. These viruses include pig lymphotropic herpesvirus (PLHV), pig cytomegalovirus (PCMV), and pig circovirus (PCV) (10, 23, 35), all of which are highly prevalent in pig populations (1, 6, 7, 36). Since PLHV is very similar to lymphoproliferative herpesviruses of other species, this virus should be included in the follow-up program for pig cell transplant recipients. In vitro experiments have shown no transmission of PCMV to human cells (37). However, data on activation of cytomegalovirus in pig-to-primate organ transplantation demonstrate that PCMV may be an important pathogen in immunosuppressed xenograft recipients (22). It has been shown that PCV type 2 can be transmitted to human cells in vitro (14). It seems prudent, therefore, to include testing for a range of pig viruses in the follow-up of transplantation patients.

The aim of this study was to look for evidence of viral infection (PERV, PCMV, PLHV, and PCV type 2) in patients that had received pig islet cell transplants.

Eighteen type 1 diabetic patients received neonatal pig islet cell transplants via three different modes of delivery: encapsulated islets (8), Sertoli islet structures in a specially designed device (38), and free islet cells (Table 1). Pig cells were obtained from pigs of Large White and Cambrough breeds that had been screened extensively for conventional microorganisms and for viruses of particular relevance to xenotransplantation, namely PLHV, PCMV, and PCV. In none of the tested donor pigs were these viruses found (12).

TABLE 1.

Antibodies against PERV proteins and pig cellular proteins detected in xenotransplantation patients

Patient no.	Transplantation date	Mode of delivery	Wk before (−) or after transplantation	Presence of antibodies against:a
				PERV
				gp70	p27Gag	p15E	Pig proteins
1	1993	Nonencapsulated islets	−214.6	−	−	−	−
			15.9	−	−	−	−
			460.3	++	−	−	+
2	1994	Nonencapsulated islets	−6.9	−	−	−	−
			2.0	−	−	−	−
			14.4	−	−	−	−
			410.0	+++	−	−	+
3	1994	Nonencapsulated islets	−0.4	−	−	−	−
			2.7	−	−	−	−
			15.4	−	−	−	−
			427.6	−	−	−	−
4	1994	Nonencapsulated islets	2.4	+	−	−	−
			163.6	+	−	−	−
			394.4	−	−	−	−
5	1996	Encapsulated islets	2.7	−	+	−	++
			44.3	−	+++	−	++
			312.0	−	++	−	+
6	1996	Encapsulated islets	−2.3	−	−	−	+
			2.6	−	−	−	+
			42.9	−	−	−	+
			311.9	−	−	−	−
7	2000	Islets + Sertoli cells	0.0	−	−	−	−
			1.1	−	−	−	+
8	2000b	Islets + Sertoli cells	−13.0	−	+	−	−
			1.9	−	++	−	−
			6.0	−	++	−	−
			19.1	−	++	−	−
9	2000	Islets + Sertoli cells	−13.0	−	−	−	−
			1.9	−	−	−	+
			5.9	−	−	−	+
10	2000	Islets + Sertoli cells	1.7	−	−	−	+
			6.6	−	−	−	+
			15.3	−	−	−	+
11	2000	Islets + Sertoli cells	−8.9	−	++	−	−
			2.7	−	++	−	+/−
			4.6	−	++	−	+/−
12	2001b	Islets + Sertoli cells	−1.1	−	−	−	−
			1.7	−	−	−	−
			6.6	−	−	−	−
			15.3	−	−	−	−
13	2001	Islets + Sertoli cells	−8.9	−	−	−	−
			2.7	−	−	−	+
			4.6	−	−	−	+
			7.6	−	−	−	+
14	2001	Islets + Sertoli cells	−6.0	−	−	−	−
			2.7	−	−	−	++

^aThe presence of antibodies is characterized as follows: −, none; +/−, none or weakly positive; +, weakly positive; ++, moderately positive; +++, strongly positive.

^bMicrochimerism (presence of pig cells) was detected on one occasion; repeated bleeding after 1 month did not show any further evidence of PERV or pig cellular marker.

Only one recipient had been immunosuppressed. This patient had previously received an allogeneic kidney transplant. All patients were given approximately 10,000 islet equivalents per kg of body weight. The Sertoli islet structures consisted of 20 to 100 Sertoli cells per islet equivalent.

To date, recipients have been monitored up to a maximum of 9 years. Recipient monitoring has included testing peripheral blood mononuclear cells (PBMC) and blood plasma for PCV 2 (19), PLHV (7), PCMV (2, 11, 13), and PERV by PCR or reverse transcription-PCR (25). This testing has been carried out on each recipient before transplantation, at nine intervals during the first year posttransplantation, and once each subsequent year. From 14 patients, serum was collected before transplantation and at several intervals after transplantation for PERV serology (Table 1).

Screening for PERV infection was performed by using Western blot analysis and purified human cell-adapted PERV as an antigen (Fig. 1). In addition, two recombinant proteins were used as antigens, the transmembrane envelope protein p15E (9) and the major core protein p27Gag (17). The sequence amplified from pig PK-15 cells corresponded to the ectodomain of the transmembrane envelope protein p15E (amino acids 488 to 597) of PERV-A. For the generation of the PERV core protein p27Gag, the entire sequence encoding this protein was amplified and cloned. Both were expressed in Escherichia coli. Several PERV-specific antisera were used as positive controls, e.g., goat antisera against the recombinant p15E, the viral p15E, the surface envelope protein gp70, and the recombinant p27Gag.

FIG. 1.

Western blot analysis of sera from pig islet cell transplant recipients (only four selected patients are shown). In the upper panel, purified virus was used as the antigen; in the lower panels, recombinant p15E (rp15E) or p27Gag (rGag) was used as the antigen. Animal sera against viral proteins were used to evaluate the quality of the blots used. Negative values indicate the number of weeks before transplantation (Tx).

In the follow-up period of up to 9 years, pig islet cell transplantation was well tolerated by all 18 type 1 diabetics. No patient was admitted to the hospital for febrile disease. No patient had signs of lymphoproliferative or neurological disease. Studies of all patients receiving porcine islet cell xenotransplantation have not shown transmission of porcine viruses. PCR and reverse transcription-PCR analysis of PBMCs and plasma showed no evidence of PERV infection in transplanted patients. PBMC DNA from two patients was found to be positive for both PERV and pig cytochrome oxidase subunit II on one occasion each. Repeated analyses of samples taken at a later time point did not show any evidence of this porcine virus or porcine cells. Analysis for PERV-specific antibodies did not show evidence for PERV transmission since none of the patient's sera reacted simultaneously against Gag and Env (Fig. 1; Table 1). Three of the 14 patients tested had antibodies against p27Gag of PERV which were already detected before treatment and which have been described also for normal blood donors (34). Antibodies against p24Gag of HIV-1 were also described for healthy uninfected persons (28). The reaction against the gp70 is certainly directed against the carbohydrate part of the molecule (18). Some of the sera reacted with pig antigens (Table 1).

All 18 patients (four patients not shown in Table 1 had also received islet and Sertoli cells) were repeatedly checked for the presence of PCV, PCMV, and PLHV DNA, and all PCR results were negative.

This study represents a long-term follow-up of human patients receiving pig islet cell xenotransplantation and further confirms the absence of transmission of PERV and of other potentially xenotic pig viruses.