Department of Microbiology, University of Minnesota, Minneapolis, MN 55455, USA1
Division of Basic Biomedical Sciences, Molecular Microbiology & Immunology Group, University of South Dakota School of Medicine, Vermillion, SD 57069, USA2
Author for correspondence: Peter Plagemann. Fax +1 612 626 0623. e-mail plag001{at}tc.umn.edu
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Abstract |
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Introduction |
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Previous studies have shown that the hypergammaglobulinaemia caused by LDV is only partly dependent on T cells. It occurs to some extent in nude and T cell-depleted mice (Li et al., 1990 ; Coutelier et al., 1990
; Hu et al., 1992
) and in the complete absence of an anti-LDV antibody response (Rowland et al., 1994
; Cafruny et al., 1999
). Thus it is not simply an indirect result of the antiviral immune response (bystander effect; Ahmed & Oldstone, 1984
). It has been shown that B cells from T cell-depleted and non-depleted mice after an LDV infection exhibit a similar elevated proliferative response in vitro, but that the former do not produce IgG2a in vitro unless treated with lipopolysaccharide and IFN-
(Coutelier et al., 1990
). Furthermore, T cells from LDV-infected mice as early as 4 days p.i. produce IFN-
much more rapidly and to higher levels in vitro after exposure to concanavalin A than T cells from uninfected mice, and IFN-
mRNA is detected in the spleen (Plagemann et al., 1995
). The results suggested that an LDV protein may function as a direct B cell mitogen and that the generation of IgG2a-producing plasma cells is then mediated by IFN-
produced by T cells largely generated in the course of the anti-LDV immune response (Plagemann et al., 1995
).
One possibility was that the B cell mitogenic response was mediated by the large N-glycans associated with the ectodomain of the primary envelope glycoprotein (VP-3) of LDV (see Fig. 1). We have therefore compared the increase in plasma IgG2a and formation of immune complexes in mice that are infected either with the two biologically cloned common quasispecies of LDV (LDV-P and LDV-vx) or with two biologically cloned laboratory mutants, LDV-C and LDV-v, that are neuropathogenic in C58 and AKR mice. In LDV-P/vx virions the VP-3P ectodomain possesses three N-glycosylation sites (Fig. 1
) that carry large N-glycan chains (Faaberg & Plagemann, 1995
). These N-glycans suppress the immunogenicity of the single LDV neutralization epitope that is located in the VP-3 ectodomain (Li et al., 1998
) and render these LDVs highly resistant to antibody neutralization (Chen et al., 2000
). The VP-3P ectodomains of the neuropathogenic LDV-C/v lack the two N-terminal N-glycosylation sites (Fig. 1
). This increases the immunogenicity of the neutralization epitope and renders these LDVs susceptible to antibody neutralization, but at the same time endows them with neuropathogenicity for C58/AKR mice (Chen et al., 1999
, 2000
). In addition, we examined IgG2a hypergammaglobulinaemia and the formation of immune complexes in mice infected with neutralization escape variants of LDV-C/v whose VP-3P ectodomains have regained two N-glycosylation sites (Fig. 1
).
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Methods |
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LDVs.
The non-neuropathogenic LDV-P and LDV-vx and the neuropathogenic LDV-C and LDV-v have been cloned out from original isolates LDV-PLA, LDV-C-BR and LDV-VIR (see Chen & Plagemann, 1997 ) by repeated end-point dilution in mice (Chen et al., 1997
, 1998
). LDV-74 is an LDV isolated from a wild house-mouse in Montana (Li et al., 2000
). Stocks of these LDVs consisted of plasma harvested from groups of mice 1 day p.i. and contained approximately 109 infectious dose (ID)50/ml. Virus titres were determined by an end-point dilution assay in FVB mice that is based on the 5- to 10-fold plasma elevation of lactate dehydrogenase activity characteristic for LDV-infected mice (Chen & Plagemann, 1997
).
Mice were infected intraperitoneally with about 106 ID50 of the LDV quasispecies indicated in appropriate experiments. In order to allow valid comparisons of mice infected with the various LDVs, we injected in general in an experiment groups of two mice with at least one neuropathogenic and one non-neuropathogenic LDV; the groups were then housed in separate cages.
Plasma IgG2a quantification.
IgG2a was quantified by capture ELISA basically as described previously (Li et al., 1990 ). In brief, 96-well ELISA plates (Pro-Bind Assay Plate, Falcon 3915; Becton Dickinson) were coated with goat anti-mouse IgG (Sigma; 2 µg/ml in bicarbonate buffer, pH 9·6; 100 µl per well). After overnight incubation at room temperature, the plates were washed three times with PBS containing 0·05% (v/v) Tween 20 (PBSTween) and then incubated for 1 h at 37 °C with PBSTween containing 1% (w/v) BSA (blocking solution; 100 µl per well). Then the plates were incubated at 37 °C for 2 h with 100 µl per well of 2-fold dilutions of standard IgG2a (Southern Biotechnology; from 1 µg/ml to 0·5 ng/ml) or six 2-fold dilutions of plasma to be assayed (from 1:4000 to 1:128000). The plates were washed a further three times with PBSTween and then incubated with alkaline phosphatase-conjugated goat anti-mouse IgG2a (Southern Biotechnology) diluted 1:1000 in blocking solution (100 µl per well). Thereafter the plates were washed three times with PBSTween and once with 50 mM TrisHCl (pH 7·4) and incubated with alkaline phosphatase substrate (Sigma 104; 200 µl per well) for 30 min. The absorbance of the reaction product was measured at 405 nm with an automatic plate reader. The concentration of IgG2a of experimental samples was estimated by extrapolation from the linear portion of the standard curve. There was no cross-reaction with other IgG isotypes (Li et al., 1990
). In general, two plates were processed at the same time allowing direct comparison of plasma samples from seven mice (four time-points each; see Fig. 2
).
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Results |
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The absolute plasma IgG2a values estimated in the different experiments varied somewhat (Fig. 2). Those of uninfected C57BL/6 mice were generally lower (<0·1 mg/ml) than those of uninfected FVB mice (0·10·5 mg/ml) and increased to a lower level than in FVB mice after an LDV infection (Fig. 2
). Similar mouse strain differences in IgG2a levels have been observed previously (Li et al., 1990
). In contrast, the observed slight variations in absolute IgG2a values for FVB mice in different experiments seem to be due to small variations (about 2-fold) in the standard IgG2a curves generated in different assays. In order to minimize such variations and to facilitate valid comparisons between the IgG2a levels in LDV-P/vx and LDV-C/v infected mice we have used the same stock solution of IgG2a for establishing the standard curves (1 µg to 0·5 ng/ml) in all assays and have simultaneously assayed the time-courses of IgG2a elevation (four time-points each; see Fig. 2
) in seven mice infected with the two classes of LDV (analysing 2-fold dilutions of plasma from 1:4000 to 1:128000).
Since LDV-v is a genetic recombinant of LDV-vx that has, by a double recombination, specifically acquired the 5' end of ORF5 of LDV-C (~400 nt), which encodes the ectodomain of the primary envelope glycoprotein VP-3P (Li et al., 1999 ), the difference in polyclonal B cell activation between LDV-P/vx, on the one hand, and LDV-C/v, on the other hand, must reside in this segment of VP-3P. The most likely molecular structure responsible for this difference in the polyclonal B cell activation is the number of N-glycans associated with the VP-3P ectodomain (Fig. 1
), since there are no other amino acid differences in this VP-3P segment that correlate with the various differences in phenotypic properties of the two classes of LDV (Chen et al., 1998
; Li et al., 1999
).
This hypothesis was further explored by examining plasma IgG2a levels in FVB mice after infection with three neutralization escape mutants of LDV-C and LDV-v (LDV-v-T105/38, LDV-C-T168/55 and LDVC-T82/68N/76) whose VP-3P ectodomains had regained three N-glycosylation sites (see Fig. 1). The plasma IgG2a elevation in duplicate mice infected with each of the three neutralization escape mutants was comparable to that in an LDV-P infected mouse and much higher than that in an LDV-C infected mouse (Fig. 3
). Comparable results were obtained in a repeat ELISA of the plasma samples; the results showed that the elevation in plasma IgG2a in the mice infected with all three neutralization escape mutants was similar to that in an LDV-vx-infected mouse and over twice as high as in an LDV-v-infected mouse (data not shown). Similarly, in a mouse infected with an LDV isolated from a wild house-mouse in Montana (LDV-74), whose VP-3P ectodomain possesses all three N-glycosylation sites (Li et al., 2000
), plasma IgG2a levels increased similarly as in LDV-P- and LDV-vx-infected mice (Fig. 2f
).
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On the other hand, immune complex formation in mice infected with the three neutralization escape mutants of LDV-C and LDV-v, whose VP-3P ectodomains had regained three N-glycosylation sites (see Fig. 1), was comparable to that in mice infected with LDV-P and LDV-vx rather than to that in mice infected with the LDV-v (or LDV-C) parent (Fig. 5
). The same was the case for immune complex formation in a mouse infected with the wild house-mouse LDV-74 (Fig. 4d
), whose VP-3P ectodomain possesses three N-glycosylation sites, just like those of LDV-P and LDV-vx.
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To further investigate the role of T and B cells in the polyclonal activation of B cells by an LDV infection, we compared the formation of plate-binding immune complexes in wild-type, nude and SCID BALB/c mice and in B cell-deficient C57BL/6 mice after infection with LDV-vx or LDV-P (Fig. 7). There was some immune complex formation in LDV-vx-infected nude BALB/c mice, but it was much lower than in LDV-vx-infected wild-type BALB/c mice (Fig. 7a
, b
), similar to that previously reported for LDV-PLA-infected nude Swiss mice and CD4+ T cell-depleted BALB/c mice and their wild-type companions (Hu et al., 1992
). The degree of reduction in immune complex formation in nude and T cell-depleted mice correlated with a similar reduction in IgG2a hypergammaglobulinaemia (data not shown; Hu et al., 1992
). In contrast, no immune complexes were detected in LDV-P-infected SCID BALB/c mice or B cell-deficient C57BL/6 mice (Fig. 7c
), and the plasmas of these mice were devoid of IgG2a (data not shown).
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Discussion |
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The finding that an LDV infection, even in CD4+ T cell-depleted mice, induces a general B cell proliferation (Coutelier et al., 1990 ) indicates that the B cell stimulation is occurring independently of T cell help. Thus, the VP-3P ectodomain with its N-glycans is a direct B cell mitogen behaving like a TI-l antigen similar to bacterial polysaccharides, lipopolysaccharides and polymeric proteins (Janeway et al., 1999
).
It is unclear, however, why the N-glycans on the VP-3P ectodomain play the major role in B cell activation, since the LDV genome encodes three other glycoproteins (Plagemann, 1996 ). ORF2 encodes a very minor envelope glycoprotein, whose about 160 amino acid long ectodomain also carries three closely spaced N-glycans (Faaberg & Plagemann, 1995
). ORF 3 encodes a soluble non-structural glycoprotein with six widely spaced N-glycosylation sites and ORF 4 encodes a non-structural, membrane-associated, glycoprotein with unknown functions, whose ectodomain possesses five N-glycosylation sites (Faaberg & Plagemann, 1997
). Perhaps the VP-3P ectodomain with its N-glycans exposed on the surface of LDV virions forms a rather rigid structure that is required for its B cell mitogenic activity. The VP-3P ectodomain is very short (about 30 amino acids long; see Fig. 1
) and linked by a disulfide bond to the even shorter (about 11 amino acids long) ectodomain of the matrix (M) protein, probably via conserved cysteine residues in the ectodomains of the two proteins (Faaberg & Plagemann, 1995
). In addition, or alternatively, B cell activation may require cross-linkage of receptor sites on the B cells, or at least multiple interactions between sites on the inducer and sites on a B cell, and such can only be accomplished by the high density of VP-3P ectodomains covering intact LDV virions. It is of interest that the polyclonal activation of B cells coincides with the accumulation of large numbers of virions in newly formed germinal centres of the spleen and lymph nodes (Anderson et al., 1995
). If the N-glycans of the VP-3P ectodomain are solely responsible for the B cell mitogenic activity of LDV, one would predict that loss of all three N-glycosylation sites on VP-3P would abolish all B cell mitogenic activity of LDV. We have not been able to test this hypothesis since we have not been able to strip LDV virions of the N-glycans without loss of infectivity and have not found an LDV mutant that lacks all N-glycosylation sites in VP-3P. Thus, we cannot rule out that some other LDV glycoprotein may contribute to the B cell mitogenic activity of LDV.
The finding that a hypergammaglobulinaemia was induced by an acute LDV infection and not by injection of glutaraldehyde-inactivated LDV (Cafruny et al., 1986a ) can be readily explained as a concentration difference. Polyclonal activation of B cells is only observed at relatively high concentrations of a TI-1 antigen (Janeway et al., 1999
), which was probably not achieved by injection of the inactivated virus, whereas an acute infection results in the production of very large amounts of progeny virions during the first day p.i. (Fig. 6
). Furthermore, inactivation of LDV by treatment with glutaraldehyde may destroy the structures on the virion surface that are critical for B cell activation as well as for the generation of neutralizing MAbs, since none of the anti-VP-3P MAbs generated to glutaraldehyde-inactivated LDV possessed neutralizing activity (Harty et al., 1987
).
Although T cell help does not seem to be required for the induction of B cell proliferation by LDV, CD4+ T cells clearly play some role in the induction of IgG2a production since both the IgG2a hypergammaglobulinaemia and immune complex formation are greatly reduced in nude mice and CD4+ T cell-depleted mice (Hu et al., 1992 ; Fig. 7
). As discussed already, it seems likely that TH1 cells generated in the course of the anti-LDV immune response of the host and induced by IL-12 produced by LDV-infected or activated macrophages (Coutelier et al., 1995
) provide the IFN-
that mediates the differentiation of the activated B cells to IgG2a-producing plasma cell. This view is further supported by the recent finding that both the IgG2a hypergammaglobulinaemia and autoantibody and immune complex formation are also reduced in C57BL/6 IFN-
gene knock-out mice, whereas LDV viraemia was the same as in wild-type C57BL/6 mice (Cafruny et al., 1999
).
Another finding that requires an explanation is that the formation of plate-binding immune complexes is reduced in LDV-C/v-infected mice, as compared to LDV-P/vx-infected mice, much more than the IgG2a hypergammaglobulinaemia (Figs 2 and 4
). A similar finding has been made in comparing the polyclonal activation of B cells in LDV-P-infected IFN-
gene knock-out and wild-type mice (Cafruny et al., 1999
). These results suggest that the increased production of polyclonal IgG2a does not necessarily lead to the generation of immune complexes containing IgG2a autoantibodies. An explanation may be derived from the fact that only a very minor fraction of the total IgG2a that is produced in LDV-infected mice is sequestered in immune complexes (Hu et al., 1992
) as well as the likelihood that the autoantibodies in these complexes are produced by a subset of B cells, such as CD5 B cells (B1 cells; Janeway et al., 1999
), that may respond differently to the mitogenic activity of LDV than the bulk of the B cells. On the other hand, it is possible that factors other than autoantibodies and their antigens are involved in the formation of the ELISA plate-binding immune complexes since we have not been able to artificially generate plate-binding immune complexes in vitro or to regenerate the immune complexes once they have been dissociated (Hu et al., 1992
).
To our knowledge, this is the first report implicating N-glycans exposed on the surface of an enveloped virus in the polyclonal activation of B cells during an acute infection. It seems likely that other enveloped viruses that cause a polyclonal activation of B cells possess similar N-glycan-containing structures associated with their envelope glycoproteins that function in a similar manner.
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Acknowledgments |
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References |
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Received 18 January 2000;
accepted 28 April 2000.
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