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Journal of General Virology (1994), 75, 1173 1176.
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1173
Difference in neutralization between lactate dehydrogenase-elevating virus
isolated from acutely and chronically infected mice
Philippe M o n t e y n e * and J e a n - P a u l Coutelier
Unit of Experimental Medicine, International Institute of Cellular and Molecular Pathology and Universitd CathoIique
de Louvain, Avenue Hippocrate 74, B-1200 Brussels, Belgium
Mouse infection with lactate dehydrogenase-elevating
virus (LDV) leads to lifelong viraemia, despite the
production of neutralizing antiviral antibodies. To test
whether viral persistence correlated with the development of resistance to these antibodies, we compared
the neutralization of viral particles derived from acutely
and chronically infected animals, using polyclonal and
monoclonal anti-LDV antibodies. Whereas virus isolated during acute infection was efficiently neutralized,
titres of LDV from chronically infected mice were only
slightly reduced by antiviral antibodies. In addition,
LDV from animals acutely infected with such poorly
neutralizable virus from chronically infected mice was
resistant to anti-LDV antibodies like their parental viral
particles. These results suggest that LDV variants
capable of escaping neutralization by antiviral antibodies
can emerge in chronically infected animals.
Lactate dehydrogenase-elevating virus (LDV), a mouse
virus related to equine arteritis virus and simian
haemorrhagic fever virus (de Vries et al., 1992; Godeny
et al., 1993; Kuo et al., 1992) and to Lelystad virus
(Meulenberg et al., 1993), induces in adult mice a lifelong viraemia, despite the production of antibodies that
have been shown to neutralize virus particles efficiently
in vitro (Cafruny et al., 1986; Coutelier et al., 1986;
Coutelier & Van Snick, 1988; Notkins et al., 1966;
Plagemann et al., 1992). Whereas anti-LDV antibodies
do not prevent in vivo virus replication in macrophages
and subsequent chronic infection, they can protect
susceptible mouse strains against virus-induced polioencephalomyelitis (Harty & Plagemann, 1990). In addition to direct neutralization, binding of anti-LDV
antibodies to virus particles may lead to the formation of
infectious complexes that can be further neutralized with
anti-mouse immunoglobulin reagents (Notkins et al.,
1968; Coutelier & Van Snick, 1988). Such a disparity in
the results of antibody-virus interactions suggested
heterogeneity in the viral population, a hypothesis also
supported by the isolation of neutralization escape
variants after serial incubations of viral particles with
anti-LDV monoclonal antibodies (Harty & Plagemann,
1988). If such variants were naturally selected in vivo
during chronic infection, this could explain, at least
partly, why viraemia does not disappear in the presence
of a well established neutralizing anti-LDV response.
To test this hypothesis, we isolated LDV during the
acute [1 day post-infection (p.i.)] and chronic (2 months
p.i.) phases of infection from five animals in a pool of 30
female 129/Sv mice (bred by G. Warnier at the Ludwig
Institute for Cancer Research, Brussels, Belgium) that
had been infected by intraperitoneal injection of 2 x 107
IDs0 of LDV (Riley strain; from the ATCC). To prepare
polyclonal antiviral antibodies, we collected the sera of
the 30 mice 2 months p.i., pooled the samples and
adjusted the pH to 8 with Tris-HCl-buffered saline. The
immunoglobulins were then extracted by anion exchange
on DEAE-Sephadex before precipitation with 40%
ammonium sulphate. Samples of LDV were purified by
centrifugation through a 2.8 ml 10 % sucrose cushion for
1 h at 40500 r.p.m, in a SW60 rotor (Beckman) and
adjusted to a concentration of approximately 105
IDs0/ml. Of these samples 25 lal was then incubated in
100 lal PBS, alone or with 10 lag anti-LDV antibodies for
15 min at 37 °C, followed by 1 h at 4 °C. Viral titres were
subsequently measured after injection of serially diluted
samples into groups of four N M R I mice and assay of
plasma lactate dehydrogenase 4 to 5 days later (Rowson
& Mahy, 1975). Whereas anti-LDV antibodies reduced
the titre of virus from acute infection by more than 104fold, they displayed only a marginal effect on the virus
derived from the same animals when chronically infected
(Fig. 1). After that, the virus derived from chronically
infected mice was transmitted into a new group of four
N M R I mice by injecting 1 lal of plasma (diluted in 500 lal
isotonic solution) into each mouse. One day later, a new
LDV sample (called here 'chronic-derived', with a titre
similar to that of the original acute virus) was prepared
from plasma from these animals and incubated with antiLDV antibodies as already described. This virus prep-
0001-1845 © 1994 SGM
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4
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3
0
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Acute
Chronic
Chronicderived
Virus isolate
Fig. l. LDV neutralization with polyclonal antiviral antibodies. LDV
samples obtained during acute (24 h p.i.) or chronic (2 months p.i.)
phases of infection of a group of 129/Sv mice, or 24 h after secondary
passage (chronic-derived) were adjusted to a concentration of approximately 105 IDs0/ml before in vitro neutralization with polyclonal
antiviral antibodies prepared from the pool of infected mice. Titres of
virus incubated with (hatched columns) or without (black columns)
antibody, ±s.o., are indicated.
aration was found to resist neutralization in a way
similar to its parental LDV isolate (Fig. 1). This chronicderived virus was again purified by centrifugation on a
sucrose cushion and transmitted to another group of
mice by injecting each animal with 500 lal of a 12 x l0 %
fold dilution of this sample. Again, we observed a
significantly lower neutralization of the chronic-derived
virus (10179-fold reduction of LDV titre) in comparison
with the neutralization of the acute virus sample
identically transmitted to another group of mice (more
than 10~-fold reduction of LDV titre).
To confirm this result, LDV samples were also
incubated with 50 gg of a pool of three different purified
monoclonal antibodies recognizing the VP3 viral envelope protein. This mixture of antibodies, at this dose,
had been previously found to be the most efficient for
0
Acute
Chronic
Chronicderived
Virus isolate
Fig. 2. LDV neutralization with monoclonal antibodies. LDV samples,
as described in Fig. 1, were neutralized with a pool of purified
monoclonal anti-VP3 IgG3 antibodies (C3904H12, C3901B3M and
C7506B12M, from BALB/c mice infected with LDV-Riley strain).
Titres of virus incubated with (hatched columns) or without (black
columns) antibody, + s.D., are indicated.
neutralization of the virus in vitro (Coutelier & Van
Snick, 1988). Again, a strong reduction in the titre of
virus isolated from acutely infected animals was observed
(Fig. 2). In contrast, both LDV derived from the same
mice at a chronic stage of infection and chronic-derived
virus obtained after secondary passage were only poorly
neutralized by this pool of monoclonal antibodies.
Since experiments with pooled reagents obtained from
groups of animals might not exactly reflect what happens
naturally in one infected host, LDV samples were derived
from individual NMRI mice 1 day and 2 months p.i., or
24 h after secondary passage of the chronic virus into
groups of four animals. Antiviral antibodies were also
prepared from the serum of the same individual mice
that were repeatedly bled after isolation of the chronic
virus. Because of the limited amount of anti-LDV
antibodies that we could obtain from these individual
animals, virus neutralization could only be compared for
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Table 1. Neutralization of LD V isolated from individual N M R I mice
LDV titre (logl0)t
Mouse
Anti-LDV
antibody
1
Polyclonal:~
2
Polyclonal
2
Monoclonal§
3
Monoclonal
4
Monoclonal
LDV
isolate*
Without
antibody
Acute
Chronic
Acute
Chronic-derived
Acute
Chronic-derived
Acute
Chronic-derived
Acute
Chronic-derived
4.38 ± 0-35
4.33 ±0.36
6.50±0.41
5'00 ± 0.53
6.50±0.31
6"33 ± 0.36
4.50±0.41
4.67 ± 0.36
4.67 ± 0-36
5.00 + 0.44
With antibody
2.33 ± 0-36
4.00±0-53
4-00±0.57
4.33 ± 0.36
< 2-50
3.67 ± 0.36
2.33±0.36
3.33 ± 0-36
2.33 ± 0.36
4.50 _+0.41
Reduction
of LDV
titre
(logx0)
2-05
0.33
250
0.67
> 4.00
2.66
2.17
1.34
2,34
0-50
* LDV isolated during acute (24 h p.i.) or chronic (2 months p.i.) phases of infection, or after
secondary passage.
t LDV infectivity titre (ID~0/ml) ± s.o.
:~ Prepared from pooled bleedings from the individually analysed mice.
§ Pool of three anti-VP3 monoclonal antibodies, as described in Fig. 2.
LDV obtained during acute infection and either virus
from chronically infected mice or chronic-derived LDV.
The results obtained with virus isolated from individual
mice were similar to those observed with pools of
animals (Table 1). LDV derived from acutely infected
mice was efficiently neutralized by antibodies produced
later by the corresponding animal, whereas virus particles
obtained when the ant±viral response was fully developed,
or the progeny of this chronic virus, escaped neutralization by the same antibody preparation. This difference
between acute virus and LDV particles obtained after
secondary infection with a chronic virus was also
observed when neutralization experiments were performed with the pool of anti-VP3 monoclonal antibodies,
however with some variations from one mouse to another
(Table 1).
Our results confirm the heterogeneity of the viral
population suggested by differences in sensitivity towards
a given preparation of antibodies (Coutelier & Van
Snick, 1988). Heterogeneity between viral isolates,
including variants that were selected after passages in
particular strains of mice, has also been demonstrated by
neutralization with rabbit anti-LDV serum (Cafruny &
Plagemann, 1982), by RNA oligonucleotide fingerprint
analysis (Brinton et al., 1986) and by nucleotide and
amino acid sequence analysis (Kuo et al., 1992). In our
study, LDV isolated early after inoculation, but not the
virus obtained later from the same animal, was efficiently
neutralized by antibodies that were secreted in response
to infection. The presence of non-neutralizing anti-LDV
antibodies in chronically infected mice leading to the
formation of infectious virus-antibody complexes (Notkins et aI., 1966) could easily explain the difference in
neutralization between acute and chronic virus. However, the observation of the persistent resistance after
passage of the poorly neutralizable chronic virus in a new
set of mice favours the hypothesis that variants of LDV
resisting neutralization are selected during the infectious
process. This hypothesis is further supported by the
observation that virus isolated after a secondary passage
remained poorly neutralizable. Indeed, it is quite unlikely
that significant amounts of ant±viral antibodies have
been transferred to the animals that received the
equivalent of 4x 10 5 lal of plasma from the initial
chronically infected mice and therefore that reduction in
neutralization of the virus purified from these animals is
related to passive immunization with non-neutralizing
anti-LDV antibodies. The emergence of variants that we
report was not strain-specific since it was observed with
129/Sv mice (Fig. 1 and 2) as well as with NMRI mice
(Table l). Therefore, our results indicate that the
selection of neutralization escape mutants, which has
been demonstrated by others after incubation with
monoclonal antibodies (Harty & Plagemann, 1988), may
happen also naturally in the course of infection in
response to ant±viral immunity. Hence this phenomenon
could contribute to some variations that have been
reported between LDV isolates in addition to other
possible mechanisms such as selection by cellular immune
responses.
Antigenic variants escaping neutralization by ant±viral
antibodies have been isolated for other viruses. Lent±viruses, like LDV, persist indefinitely in their hosts,
replicate continuously during the lifelong infection and
undergo antigenic variations in the presence of neutralizing antibodies. The development of an animal model in
which a standard strain of equine infectious anaemia
virus induces a chronic disease allowed the characterization of viral antigenic variations and the study of
genomic alterations that were associated with this
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persistent infection (Payne et al., 1984). The analysis of
visna virus mutants appearing during persistent infection
of sheep showed a small number of changes in their
oligonucleotide pattern (Clements et al., 1980). Variations were also detected in the genome of human
immunodeficiency virus isolates derived from persistently
infected individuals (Hahn et al., 1986).
A role, if any, of anti-LDV antibodies in controlling
virus replication is still to be demonstrated. It has been
shown that LDV titres fluctuate because of limited
availability of permissive cells rather than because of the
immune response. For example, immunodeficient nude
mice develop a viraemia comparable to that of their
normal counterpart (Onyekaba et al., 1989). The selection of antibody-resisting LDV variants in immunocompetent animals is compatible with this similarity in
viral titres. Indeed, it can be postulated that in the
absence of escape mechanisms anti-LDV antibodies
would be capable of eliminating the virus. The role of
naturally occurring neutralization escape variants in
persistent viraemia, as well as the mechanisms by which
these viruses resist neutralization remain to be determined. Such an analysis would be greatly facilitated
by the development of a cell line allowing LDV cloning
and amplification and by sequence analysis of isolated
variants.
We are indebted to J. Van Snick and to P. L. Masson for critical
reading of the manuscript. This work was supported by the Fonds
National de la Recherche Scientifique (FNRS), Fonds de la Recherche
Scientifique M6dicale (FRSM), Loterie Nationale, Op6ration T616vie,
the State Prime Minister's Office Science Policy Programming (interuniversity attracting poles, grant 44), and the French Community
(concerted actions, grant 88/93-122), Belgium. P. Monteyne is research
assistant and J.-P. Coutelier is research associate with the FNRS,
Belgium.
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(Received 21 May 1993; Accepted 1 December 1993)