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Journal of Virology, February 2002, p. 1999-2002, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1999-2002.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Human CD59 Incorporation into Porcine Endogenous Retrovirus Particles: Implications for the Use of Transgenic Pigs for Xenotransplantation

Daniel M. Takefman,1 Gregory T. Spear,2 Mohammed Saifuddin,2 and Carolyn A. Wilson1*

Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892,1 Department of Immunology/Microbiology, Rush University, Chicago, Illinois 606122

Received 30 July 2001/ Accepted 15 November 2001


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ABSTRACT
 
Transgenic pigs have been engineered to express human CD59 (hCD59) in order to suppress hyperacute rejection of xenotransplants in human recipients. In this study, porcine endogenous retrovirus (PERV) was produced in a porcine cell line expressing hCD59 in order to examine the effect of this complement control protein on PERV neutralization by human sera. hCD59 was found to be incorporated into PERV particles produced from engineered ST-IOWA cells. PERV incorporation of hCD59 resulted in a dramatic inhibition of complement-mediated virolysis by human serum. However, incorporation of hCD59 had no effect on neutralization of PERV by human serum, as measured in infectivity assays. Our results suggest that the use of organs from hCD59 transgenic pigs will inhibit complement-mediated virolysis, but will not compromise the protective effects of human sera on the neutralization of PERV particles.


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TEXT
 
In order to relieve the shortage of human organs for transplantation, cells and organs of porcine origin are under consideration as an alternative. One major hurdle to xenotransplantation has been hyperacute immunologic rejection of the porcine cells. Hyperacute rejection (HAR) of porcine organs occurs rapidly and is mediated by naturally occurring human antibodies that activate the complement system. These antibodies are specific for the Gal{alpha} 1-3Gal ({alpha}-Gal) sugars that are linked to surface glycolipids and glycoproteins expressed on porcine cells (6). Unlike most mammalian cells, humans, apes, and Old World monkeys have an inactivating mutation within the gene encoding the cellular enzyme {alpha} 1,3-galactosyltransferase required to synthesize {alpha}-Gal (5). Therefore, humans, apes, and Old World monkeys do not express {alpha}-Gal, but, presumably due to environmental exposure, do produce anti-{alpha}-Gal antibodies (5).

One strategy developed to prevent organ rejection is to suppress complement activation on the {alpha}-Gal-expressing porcine cells. Transgenic pigs have been engineered that express human complement regulatory proteins (CRPs), such as decay-accelerating factor (DAF; CD55) and membrane inhibitor of reactive lysis (MIRL; CD59) (4, 9). DAF accelerates the decay of C3 and C5 convertases, while MIRL prevents assembly of the membrane attack complex (MAC). Pig organs from animals transgenic for human CRPs have shown improved survival when transplanted into nonhuman primates (2, 3, 9, 12, 28).

Transmission of infectious agents represents another potential risk associated with xenotransplantation. While it may be possible to develop herds of pigs free of known exogenous infectious agents of concern, it is currently not possible to remove endogenous retroviruses, such as the gammaretrovirus porcine endogenous retrovirus (PERV) (1). The pig genome has been estimated to carry at least 50 proviral copies of PERV, some of which are able to infect human cells in vitro (7, 11, 24, 26). Thus, it is important to consider ways in which transmission of infectious PERV can be prevented. An immune defense mechanism against PERV likely to be important is inactivation of gammaretrovirus particles that carry the {alpha}-Gal sugar (14). Gammaretrovirus neutralization by human sera is mediated through binding of {alpha}-Gal-specific antibodies to epitopes on the viral surface glycoprotein followed by activation of the complement pathway (13, 22). In a study by Patience et al. (11), human sera lysed PERV produced in porcine cells. Therefore, the same immune response that rejects xenotransplanted organs may also protect humans and Old World monkeys from gammaretrovirus infection.

One consequence of producing CD55 and/or CD59 transgenic pigs is that PERV particles may acquire these human host cell proteins on the viral membrane during the budding process, rendering the particles resistant to complement-mediated inactivation by human sera (25). Studies have shown that human immunodeficiency virus type 1 and human T-cell leukemia virus type 1 can acquire host cell CD55 and CD59 proteins at levels that protect from virolysis (16-19). As has been demonstrated, cells from CD59 transgenic pigs show decreased sensitivity to human sera (4). Here we report our analysis of the sensitivity to human sera of PERV particles produced in porcine cells that express the human CRP CD59.

Porcine cells expressing human CD59 (hCD59) were used to examine the effects of this CRP on PERV neutralization by human sera. The cDNA for hCD59 (16) was digested with BamHI and ligated into the BamHI site of the retroviral vector pLXSN (10). The resulting plasmid, pLCD59SN, was cotransfected with a Moloney murine leukemia virus (Mo-MuLV) gag-pol and a vesicular stomatitis virus (VSV-G) expression vector into 293T cells (obtained from M. B. Eiden, National Institutes of Health) to generate VSV-G-pseudotyped retroviral particles as described elsewhere (23). These particles were used to introduce the hCD59 cDNA into the porcine cell line ST-IOWA (obtained from R. Fister, Tufts University, Boston, Mass.). Following 14 days of selection with 800 µg of G418 per ml (Life Technologies, Rockville, Md.), pooled populations of G418-resistant cells were incubated with a phycoerythrin-conjugated anti-hCD59 monoclonal antibody (Becton Dickinson, San Jose, Calif.) and sorted for high-hCD59-expressing cells by using a FACSTARPlus flow cytometer (Becton Dickinson). As shown in Fig. 1A, ST-IOWA cells do not express hCD59, while the transduced ST-IOWA (hCD59-ST-IOWA) cells express high levels of hCD59. The hCD59-ST-IOWA cells were subsequently infected with PERV-NIH under conditions previously reported (20).



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  		FIG. 1. Measurement of hCD59 on porcine cells and PERV particles. (A) Fluorescence-activated cell sorter analysis of ST-IOWA cells transduced with an hCD59 expression vector (opaque histogram) and nontransduced cells (outlined histogram) stained with a phycoerythrin-conjugated antibody to hCD59 (Becton Dickinson). (B) Immunocapture of PERV by S. aureus cells was performed as described in the text. Virus from PERV-infected ST-IOWA cells (ST-PERV) or hCD59-ST-IOWA cells (hCD59-ST-PERV) was incubated with either anti-hCD59- or anti-IgG-bound S. aureus cells in duplicate tubes. The amount of captured PERV was quantified by TM-PERT (8), and each sample was assayed in triplicate. Captured PERV is shown as a percentage of total PERV added to the S. aureus cells. The data shown are the average of three experiments, with error bars representing the standard deviation.

In order to show that virus produced from hCD59-ST-IOWA cells (hCD59-ST-PERV) carries the hCD59 host cell protein, an immunocapture assay using protein A-expressing Staphylococcus aureus cells was performed as described elsewhere (15) with the following modifications. Fifty microliters of S. aureus cells (Pansorbin; Calbiochem, La Jolla, Calif.) was incubated with 25 µg of rabbit anti-rat polyclonal antisera (catalog no. 312-005-046; Jackson Immunoresearch, West Grove, Pa.), washed with phosphate-buffered saline containing 1% bovine serum albumin and then incubated with either 2 µg of rat anti-hCD59 monoclonal antibody (Biosource International, Camarillo, Calif.) or 10 µg of purified rat immunoglobulin G (IgG) (Sigma, Saint Louis, Mo.). Virus-containing culture supernatants were collected and ultracentrifuged over 20% sucrose as previously described (20), and purified virus was incubated with the S. aureus cells. Both total and bound PERV were quantified by a product-enhanced reverse transcriptase (RT) assay that utilizes an internal TaqMan probe (TM-PERT) (8). As shown in Fig. 1B, approximately 57% of hCD59-ST-PERV particles were captured with the anti-hCD59 antibody compared to less than 1% of ST-PERV particles captured with the same anti-hCD59 antibody. These results demonstrate that PERV produced from hCD59-ST-IOWA cells carries the human CD59 protein.

In order to show that the hCD59 protein carried on PERV is functional, the susceptibility of virus to complement-mediated lysis (CML) was examined. Virus-containing supernatants were ultracentrifuged. The pelleted material was resuspended in Dulbecco's modified Eagle's medium and then incubated for 1 h at 37°C with a 1:10 dilution of either blood group AB+ human serum (used as a source of both anti-{alpha}-Gal antibodies and complement) or serum that had been heat inactivated (HI) to destroy complement activity. A 1:30 dilution of serum was used because no complement lysis was observed with either PERV-ST or CD59-ST-PERV (data not shown). Virolysis was measured by RT enzyme release in a conventional RT assay (26), with the modification that serum-treated samples were incubated with RT buffer containing no detergent, while mock-treated samples were incubated with 1% Triton X-100-containing buffer to determine the 100% lysis value. As seen in Fig. 2, 21% RT release was measured from hCD59-ST-PERV, while 93% RT release was seen with PERV from ST-IOWA cells following serum treatment. Thus, the PERV particles from hCD59-ST-IOWA cells incorporate functional hCD59 at levels that protect from CML.



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  		FIG. 2. PERV-associated hCD59 inhibits CML. Sensitivity of ST-PERV and hCD59-ST-PERV to CML by human serum was assessed by measuring release of RT enzyme after treatment with either complement active human serum, complement HI serum, or Triton X-100. A 1:10 dilution of serum was used in these experiments. The amount of RT release measured after 1% Triton X-100 serves as the 100% RT release control. Due to the expression of endogenous RT activity by ST-IOWA cells, the percentage of RT release was calculated with the formula [(complement treatment - HI complement treatment)/(Triton X-100 treatment - HI complement treatment)]. Each treatment was performed in duplicate tubes. The data are shown are the average of three experiments, with error bars representing the standard deviation.

The ability of hCD59 to alter infectivity in the presence of human serum was also examined. PERV-NIH-infected human embryonic kidney 293 cells (ATCC CCL-121), ST-IOWA, and hCD59-ST-IOWA cells were transduced with a VSV-G-pseudotyped retroviral vector containing a Mo-MuLV-based genome encoding ß-galactosidase (23). This modification allowed for determination of infectivity titers of the PERV-NIH pseudotypes containing this genome by histochemical assay for ß-galactosidase activity (27). Virus-containing culture supernatants were incubated with AB+ serum, HI serum, or medium at 37°C for 1 h. Following incubation, supernatants were added to 293T cells in the presence of 6 µg of Polybrene per ml. Infectivity titers were determined 48 h postexposure to viral supernatants and then normalized to the medium-treated virus (100% control). As shown in Fig. 3, infectivity of ST-PERV was almost completely neutralized by human serum (9.6% of medium control), while as expected, 293-PERV was resistant to neutralization (86.4% of the medium control). Interestingly, hCD59-ST-PERV was also neutralized by human serum (12.6% of medium control). In all three viral preparations, the addition of HI serum had no effect on viral titer, demonstrating that {alpha}-Gal antibodies do not directly neutralize PERV in the absence of active complement. Together, the data in Fig. 3 show that incorporation of hCD59 on PERV particles is not sufficient to prevent neutralization of PERV by human serum.



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  		FIG. 3. Neutralization of PERV by human serum. In order to assess the effect of human serum on PERV infectivity, culture supernatants were collected from PERV-NIH-infected 293 (293-PERV), ST-IOWA (ST-PERV), or hCD59-ST-IOWA (hCD59-ST-PERV) cells that carried a Mo-MuLV-based retroviral vector encoding ß-galactosidase (23). Each supernatant was incubated with complement active serum, HI serum, or medium. A 1:10 dilution of serum was used in these experiments. The infectivity titers were measured by counting blue-forming units (BFU) after histochemical staining for ß-galactosidase expression (27). The number of BFU counted after exposure to medium-treated virus serves as the 100% control. All other titers are normalized to that value. Each experiment was done in triplicate, and the data are shown as the average of two experiments, with error bars representing the standard deviation. Average titers for the medium-treated controls were 221, 325, and 293 BFU for 293-PERV, ST-PERV, and hCD59-ST-PERV, respectively.

Since the function of CD59 is to inhibit MAC formation, one explanation of the neutralization results is that MAC formation is not necessary for PERV neutralization. In a study by Takeuchi et al. (21), gammaretroviruses produced in {alpha}-Gal-expressing cell lines were neutralized by human sera deficient in complement components essential for MAC formation (C7 and C9), but not human sera deficient in an upstream early complement pathway component (C2). The finding that hCD59 does not affect neutralization of PERV by human sera is consistent with the previous study by Takeuchi et al. (21). Together, these studies suggest that MAC formation is not necessary for the neutralization of gammaretroviruses by complement-activating {alpha}-Gal antibodies. Instead, neutralization of gammaretroviruses may occur by coating of the virion particles by C3b and/or C5b complement fragments that would block virus-receptor interactions. In contrast, since CD55 inhibits C3b deposition, it is likely that PERV produced from CD55-expressing cells will result in virions that are resistant to neutralization by human sera. This hypothesis will be the subject of future investigations.

In conclusion, while hCD59 is incorporated into PERV particles and is able to inhibit complement-mediated virolysis, there is no effect of this CRP alone on the ability of human serum to neutralize PERV infectivity. Use of hCD59 transgenic pigs for xenotransplantation may provide the needed balance of protection of the xenograft from HAR without compromising the protective effect of human serum neutralization of retroviral particles.


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ACKNOWLEDGMENTS
 
We thank Howard Mostowski for assistance with cell sorting. We also thank Suzanne Epstein and Andrew Byrnes for critical review of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Building 29B, Room 2E12, 8800 Rockville Pike, Bethesda, MD 20892. Phone: (301) 827-0481. Fax: (301) 827-0449. E-mail: wilsonc{at}cber.fda.gov. Back


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Journal of Virology, February 2002, p. 1999-2002, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1999-2002.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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