Institut für Virologie und Immunbiologie, Versbacher Str. 7, D-97078 Würzburg, Germany1
Nextran Inc, 303B College Road East, Princeton Forrestal Center, Princeton, NJ 08540, USA2
Author for correspondence: Jürgen Schneider-Schaulies. Fax +49 931 2013934. e-mail jss{at}vim.uni-wuerzburg.de
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Abstract |
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Introduction |
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In addition to its function in the inactivation of human complement, CD46 acts as a receptor mediating infection of cells with human herpesvirus-6 (HHV-6; Santoro et al., 1999 ) and measles virus (MV) vaccine strains (Buchholz et al., 1996
, 1997
; Dörig et al., 1993
; Gerlier et al., 1994
; Hsu et al., 1997
; Naniche et al., 1993a
; Maisner et al., 1994
; Manchester et al., 1994
, 1995
, 1997
). Infection of cells with such MV strains or contact of cells with viral glycoproteins expressed on infected cells leads to downregulation of CD46 from the cell surface of the contacted cell (Naniche et al., 1993b
; Krantic et al., 1995
; Schneider-Schaulies et al., 1996
) and renders these cells susceptible to lysis by complement in vitro (Schnorr et al., 1995
). It is not clear what effect this may have in vivo, since most cells express other complement regulatory proteins, which will serve to protect the cells from complement-mediated lysis. However, it is formally possible in humans that MV infection may contribute to a rapid clearing of CD46-negative cells by complement, and thus to the attenuation of CD46-downregulating MV strains.
Not all MV strains have the capacity to downregulate CD46 and the degree of receptor modulation varies considerably between various MV strains which use CD46 as a receptor (Schneider-Schaulies et al., 1995a , b
). A few defined amino acids in the MV haemagglutinin (H) protein determine the capacity to downregulate CD46 (Bartz et al., 1996
; Lecouturier et al., 1996
). Recently, we and others have found that a number of MV wild-type (WT) isolates appear to use receptors other than CD46 and these strains also do not downregulate human CD46 (Bartz et al., 1998
; Hsu et al., 1998
; Tanaka et al., 1998
). Interestingly, it has been found that other morbilliviruses which are closely related to MV, such as rinderpest virus (RPV), can induce the downregulation of CD46 from the surface of cells, while not using CD46 as a receptor (Galbraith et al., 1998
). Thus, receptor usage and CD46 downregulation appear to be independent processes. Recently, the pig homologue of human CD46 was cloned and found to share a similar structure and 42% amino acid identity with human CD46 (Seya et al., 1998
; Toyomura et al., 1997
; Van den Berg et al., 1997
). It is not known whether pig CD46 interacts with any strain of MV.
In this publication, we compared the susceptibility of peripheral blood mononuclear cells (PBMC) from human CD46-tg and non-tg pigs to MV infection. Since human CD46 protects non-human cells from lysis by human complement, it is important to know under which conditions CD46 may be downregulated from the surface of xenotransplants. Furthermore, the possibility exists that expression of human CD46 on tg pig tissues may render these cells susceptible to MV infection. We found that PBMC from tg as well as non-tg pigs were susceptible to MV vaccine strains and that infection resulted in downregulation of both porcine and human CD46.
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Methods |
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Isolation and culture of PBMC.
PBMC from pigs and human blood donors were isolated by centrifugation of blood cells on a Percoll cushion (density 1·123) for 20 min at 1000 g. Cells floating on the cushion were washed with PBS and frozen (1x107 per ml) in RPMI 1640 medium containing 50% foetal bovine serum (FBS) and 8% DMSO in liquid nitrogen. For the tests, cells were thawed and cultured in RPMI 1640 medium containing 10% FBS, penicillin and streptomycin. Pig PBMC were stimulated with 10 µg/ml phytohaemagglutinin (PHA) and human PBMC were stimulated with 5 µg/ml PHA.
MV strains.
The MV strains genetically belonging to the vaccine group were Edmonston (Edm), Schwarz and Moraten. These strains were propagated using Vero cells and use CD46 as a receptor on human cells and downregulate CD46 after infection or contact with infected cells (Schneider-Schaulies et al., 1995a , b
, 1996
). WT strains TC5679, Wü7671, Wü5404 and Wü4797 were isolated in 1996 from measles patients in Würzburg, Germany. The WT MV strains were isolated and propagated using the human B cell line BJAB, which was immortalized with EpsteinBarr virus (EBV) and does not produce EBV (Menezes et al., 1975
).
Antibodies.
The MAbs against CD46, MV H protein and MV nucleocapsid (N) protein (13/42, L77 and F227, respectively) were grown and purified over protein G columns in our laboratory, and used at a dilution of 5 µg/ml. FITC- or phycoerythrin-conjugated rabbit anti-mouse Ig, and goat anti-rabbitFITC were purchased from Dako and used at 1:100 dilution. Polyclonal rabbit anti-CD46 sera were a generous gift from F. Wild, (Lyon, France) and G. Yeh (Cytomed Inc, USA).
Flow cytometry.
For flow cytometry, PBMC (1x105 per tube) were harvested and washed with FACS buffer (calcium- and magnesium-free PBS containing 0·4% BSA and 0·01 M NaN3). After incubation with the first antibody diluted in FACS buffer on ice for 45 min, cells were washed twice with FACS buffer, incubated for 45 min with the second antibody, washed twice and analysed with a FACScan flow cytometer (Becton Dickinson).
Virus binding assay.
Similar m.o.i. of virus preparations of the different MV strains were used in viruscell binding assays as described previously (Bartz et al., 1998 ). The m.o.i. were determined according to titration of MV vaccine strains on Vero cells and MV WT strains on BJAB cells. PBMC (2x104) in 100 µl PBS were incubated at 4 °C for 1 h with virus at a given m.o.i., washed with FACS buffer, stained with anti-MV H protein MAb L77 and FITC-conjugated goat anti-mouse antibodies as described, and bound virus was determined by analysis with a FACscan (Becton Dickinson).
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Results |
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Discussion |
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In addition to the fact that not all MV strains downregulate CD46, it was found that the H proteins of RPV and peste-des-petits-ruminants virus, closely related morbilliviruses, also induce the downregulation of CD46 from the surface of Vero and B95a cells, although CD46 did not appear to be the cellular receptor for these viruses (Galbraith et al., 1998 ). We also observed that on chimeric recombinants, the first domain of CD46 alone is sufficient to induce downregulation, while these molecules do not serve as receptors for MV (Buchholz et al., 1996
; Firsching et al., 1999
). This indicates that CD46 downregulation and receptor usage by different morbilliviruses are distinct processes. Interestingly, another virus that uses CD46 as receptor, HHV-6, also induces its downregulation from the surface of infected cells (Santoro et al., 1999
).
Since CD46 acts in vivo to protect cells from autologous complement-mediated lysis, CD46 modulation may result in the interaction of complement cascade proteins with such cells and may induce their elimination. As we demonstrated, infection with MV can induce the downregulation of tg and endogenous CD46 from the surface of porcine cells. The pig homologue of human CD46 was recently cloned, and shares a similar structure and 42% amino acid identity with human CD46 (Seya et al., 1998 ; Toyomura et al., 1997
; Van den Berg et al., 1997
). Pig CD46 is expressed on all pig blood leukocytes and erythrocytes, and on endothelial and epithelial cell lines (Toyomura et al., 1997
). Our data suggest that CD46 downregulation after interaction with MV may also occur in porcine organs which express endogenous and/or human CD46 as a means of protection against complement-mediated damage.
There is no doubt that pig PBMC, in the absence or presence of human CD46, can be infected with MV vaccine strains. The lack of a clear inhibition of MV infection by polyclonal antibodies to CD46, which also recognize endogenous pig CD46, did not resolve the question of receptor usage, since polyclonal sera to CD46 inhibit also the infection of human PBMC only partially. The MV vaccine strains therefore seem to recognize additional receptors that are not identical to CD46 on PBMC of human as well as porcine origin. This is in contrast to Vero cells, where anti-CD46 sera efficiently inhibit the infection. The uptake mechanism of MV by PBMC requires further investigation.
The use of pigs as potential organ donors for humans has drawn attention to the potential transmission of viruses from these xenografts to recipients (Patience et al., 1997 ). Additionally, the possibility formally exists that expression of human proteins in tg pigs may result in pigs which may now acquire infections to which they were previously immune. CD46 is also involved in the adherence of group A Streptococcus pyogenes, and Neisseria gonorrhoeae and Neisseria meningitidis to cells (Atkinson et al., 1994
; Kallstrom et al., 1997
; Okada et al., 1995
). Another complement regulatory protein, CD55 (DAF), is a receptor for some strains of Escherichia coli (Nowicki et al., 1993
) and for several enteroviruses including types of echovirus (Bergelson et al., 1994
; Ward et al., 1994
) and coxsackie B virus (Bergelson et al., 1995
). Data presented here demonstrate that pigs are inherently susceptible to infection by MV vaccine strains, regardless of their tg status. Interestingly, MV WT strains, which have been described as using different receptors, did not infect pig PBMC regardless of whether they expressed human CD46. Therefore, in considering a potential xenograft recipient, it would appear unlikely that infection by WT MV would affect human CD46 expression on a tg porcine organ. Additionally, a high percentage of adult human recipients have a lifelong immunity against measles as result of vaccination or disease. Since expression of human CD46 on tg porcine cells does not alter their susceptibility to MV infection, it would therefore not appear to present an additional risk to a potential xenograft recipient in terms of MV infection.
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Acknowledgments |
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References |
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Received 13 December 1999;
accepted 29 February 2000.