Download Pseudorabies virus (PRV)-specific antibodies suppress intracellular

Journal of General Virology (2003), 84, 2969–2973
Short
Communication
DOI 10.1099/vir.0.19281-0
Pseudorabies virus (PRV)-specific antibodies
suppress intracellular viral protein levels in
PRV-infected monocytes
Herman W. Favoreel,1 Gerlinde R. Van de Walle,1 Hans J. Nauwynck,1
Thomas C. Mettenleiter2 and Maurice B. Pensaert1
Correspondence
Hans J. Nauwynck
[email protected]
Received 9 April 2003
Accepted 10 July 2003
1
Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133,
Merelbeke 9820, Belgium
2
Federal Research Centre for Virus Diseases of Animals, Insel Riems, Germany
Blood monocytes infected with pseudorabies virus (PRV), a swine alphaherpesvirus, are not
eliminated efficiently by antibody-dependent immunity and may occasionally transport PRV to the
pregnant uterus of vaccinated animals. This study examines in vitro the long-term fate of
PRV-infected monocytes cultivated in the presence of porcine PRV-specific antibodies. All
monocytes were infected and expressed viral late proteins, and 30 % of PRV-infected monocytes
cultivated with PRV-specific antibodies survived up to 194 h post-infection (p.i.), the end of the
experiment (compared to 0 % for cells cultivated with PRV-negative antibodies). Of these surviving
cells, ±75 % no longer expressed microscopically detectable viral late proteins from 144 h p.i.
onwards. Remarkably, monocytes infected with a PRV gB-null virus did not survive in the presence
of PRV-specific antibodies. These data suggest that PRV-specific antibodies suppress viral
protein levels in infected monocytes, perhaps helping the virus to persist and reach internal organs
in vaccinated animals.
The alphaherpesvirus pseudorabies virus (PRV) causes
Aujeszky’s disease in its natural host, the pig. The disease is
characterized by nervous signs in newborn pigs, respiratory
disorders in fattening pigs and reproductive failure in sows
(Wittmann et al., 1980). Abortion can be an important
consequence of PRV infection in susceptible pregnant
sows (Pensaert & Kluge, 1989). Even in the presence of
vaccination-induced immunity, PRV inoculation may
result in infection of the respiratory tract, involving
mononuclear cells in draining lymph nodes. These cells
can enter the bloodstream, resulting in a restricted viraemia
(Wittmann et al., 1980), which, in general, does not cause
problems. However, in rare instances, abortion in immune
animals can occur as a result of transplacental spread and
intrafoetal replication. Infected blood monocytes are
essential to transport the virus in vaccination-immune
pigs to enable the virus to reach the pregnant uterus
(Nauwynck & Pensaert, 1992, 1995a). Different components
of immunity, including antibody-dependent immune
effectors, should be able to lyse PRV-infected monocytes
in the blood stream of vaccinated animals. Indeed, PRVinfected monocytes express viral proteins in their plasma
membrane (Favoreel et al., 1999b) and binding of antibodies to these antigens should induce antibody-dependent
cell lysis (Sissons & Oldstone, 1980). One of the mechanisms that may aid PRV in masking infected blood monocytes from antibody-dependent cell lysis consists of the
0001-9281 G 2003 SGM
internalization of antibody–antigen complexes (Favoreel
et al., 1999b). During this process, binding of PRV-specific
antibodies to viral cell surface proteins in PRV-infected
monocytes results in rapid internalization of antibody–
antigen complexes (immune-masked monocytes). This
internalization process is fast and efficient, is mediated by
viral proteins gB and gD and has been shown to reduce the
sensitivity of PRV-infected monocytes towards antibodydependent, complement-mediated cell lysis in vitro (Van de
Walle et al., 2001, 2003a; Favoreel et al., 2002). Such
immune-masked monocytes have already been shown
in vitro to be capable of transmitting virus to vascular
endothelial cells via direct cell-to-cell spread in the presence
of neutralizing antibodies as a possible first step for PRV
to reach internal organs (Van de Walle et al., 2003b).
To date, antibody-induced internalization of viral cell
surface proteins has only been studied in vitro over relatively
short periods post antibody addition (¡2 h) and post
inoculation (¡17 h). However, the circulation of PRVinfected monocytes in vivo occurs in the continuous
presence of PRV-specific antibodies. Therefore, the aim of
the current study was to investigate the effect of the
continuous presence of porcine PRV-specific antibodies on
PRV-infected monocytes over an extended period of time,
examining both the viability of the infected cells as well as
expression level of viral proteins in infected cells.
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2969
H. W. Favoreel and others
Pigs from a PRV-negative farm were used as blood donors.
Blood was collected from the vena jugularis on heparin
(15 U ml21) (Leo) and monocytes were isolated, seeded
onto 4-well multidish plates (Nunc) and inoculated with
PRV at an m.o.i. of 10, all as described before (Van de Walle
et al., 2003b). At 1 h post-inoculation (p.i.), cells were
washed three times with RPMI 160 (Gibco-BRL) and
incubated in medium supplemented with porcine PRVspecific or PRV-negative antibodies (0?4 mg IgG ml21).
PRV-specific protein A-purified IgG antibodies were
derived, as described earlier, from a PRV (89V87)inoculated pig originating from a PRV-negative farm
(Nauwynck & Pensaert, 1995b), had an antibody titre of
512, as determined with a serum neutralization (SN) test
(Andries et al., 1978), and were able to neutralize all input
virus at the concentration used in our experiments. PRVnegative protein A-purified IgG antibodies were derived
from a PRV-negative pig and had an antibody titre of <2
with the SN test.
First, we determined the percentage of monocytes initiating
expression of viral proteins upon PRV infection. Therefore,
cells were acetone-fixed at different time-points p.i., stained
with FITC-labelled porcine PRV-specific polyclonal antibodies (Van de Walle et al., 2003b), analysed and scored
by fluorescence microscopy (Leica DM RBE microscope,
Leica). Polyclonal antibodies were derived from hyperimmune serum of a PRV-vaccinated and -infected pig
(Nauwynck & Pensaert, 1995b) and have high reactivity
against PRV late proteins gB, gD and gE (data not shown).
Fig. 1 shows that PRV inoculation at a high m.o.i. resulted
in the microscopically detectable expression of viral antigens in 100 % of monocytes at 96 h p.i. both if monocytes
were cultivated in the presence of either PRV-specific or
PRV-negative antibodies. Hence, all monocytes initiated
the expression of PRV proteins irrespective of the addition
of PRV-specific antibodies to the medium at 1 h p.i.
Then, we evaluated how long swine blood monocytes
survive a PRV infection if they are cultivated in the
continuous presence of PRV-specific or PRV-negative
antibodies. Therefore, at different time-points p.i. with
PRV at an m.o.i. of 10 (0, 24, 48, 72, 96, 120 and 196 h p.i.),
monocytes were incubated for 30 min on ice with ethidium mono-azide bromide (EMA) (Molecular Probes)
(100 mg ml21), which specifically stains the nuclei of dead
cells (red), followed by acetone fixation and staining of all
nuclei with Hoechst 33342 (Molecular Probes) for 10 min at
room temperature (blue). Fig. 2 shows that the viability of
PRV-infected monocytes decreased dramatically until 72 h
p.i., in the presence of both PRV-specific and PRV-negative
antibodies. However, from 120 h p.i. onwards, all PRVinfected monocytes incubated with PRV-negative IgG
antibodies were lysed, whereas approximately 30 % of
PRV-infected monocytes incubated with PRV-specific
polyclonal IgG antibodies remained viable (Fig. 2, box).
These results indicate that in the continuous presence of
PRV-specific antibodies, a subpopulation of PRV-infected
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Fig. 1. Efficiency of PRV inoculation. Monocytes (n=106) were
inoculated with PRV and, at 1 h p.i., supplemented with
0?4 mg ml”1 of either PRV-specific polyclonal IgG antibodies
(%) or PRV-negative IgG antibodies (n). Mock-infected monocytes incubated with either PRV-specific (e) or PRV-negative
(#) IgG antibodies served as controls. At different time-points
p.i., expression of viral antigens was determined by fixation of
cells for 20 min at ”20 6C in 100 % acetone followed by incubation for 1 h at 37 6C with FITC-labelled PRV-specific porcine
polyclonal antibodies. Data represent the means ±SD of triplicate assays.
Fig. 2. Long-term survival of PRV-infected monocytes.
Monocytes (n=106), at 1 h p.i. with PRV, were incubated with
0?4 mg ml”1 of either PRV-specific polyclonal IgG antibodies
(%) or PRV-negative IgG antibodies (n). Mock-infected monocytes incubated with either PRV-specific (e) or PRV-negative
(#) IgG antibodies served as controls. The dotted line shows
results for monocytes inoculated with a PRV gB-null virus and
incubated with 0?4 mg PRV-specific IgG ml”1. At different
time-points p.i., viability was determined by incubating the
cells for 30 min on ice with EMA to stain dead cells and,
after fixation in 100 % acetone, cells were stained by incubating
for 10 min at room temperature with Hoechst 33342 to
detect all nuclei. Data represent the means ±SD of triplicate
assays.
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Journal of General Virology 84
Antibody-induced decrease of PRV protein expression
monocytes remains viable for unusually long periods of
time. Interestingly, the processes of antibody-induced
internalization of antibody–antigen complexes that we
described earlier and the antibody-induced survival of
PRV-infected monocytes that we describe here may perhaps
be related, since the viral protein gB seems to play crucial
roles during both processes. Indeed, gB is necessary for
efficient internalization of antibody–antigen complexes
(Van de Walle et al., 2001) and, as shown in Fig. 2,
monocytes infected with a gB-deleted PRV strain (grown
on gB-complementing cells) cannot survive a PRV infection in the presence of PRV-specific antibodies (Fig. 2,
dotted line); the gB-null virus and gB-complementing
cells were described earlier (Rauh & Mettenleiter, 1991).
This indicates that antibody binding to gB may be a key
event in initiating internalization of antibody–antigen
complexes as well as in allowing a subpopulation of
monocytes to survive a PRV infection for extended
periods of time.
Next, we evaluated microscopically the expression of viral
antigens in the subpopulation of PRV-infected monocytes
that survived for unusually long periods of time in the
presence of PRV-specific antibodies (Fig. 2, box). At
different times p.i., monocytes were incubated with EMA
to stain the nuclei of dead cells (red), as described above.
Cells were then acetone-fixed and stained to detect viral
antigens (anti-PRV-FITC, green) and all nuclei (Hoechst
33342, blue), both as described above. Fig. 3(A) shows
that the expression of viral antigens in the majority of
these surviving PRV-infected cells decreased gradually.
All of the surviving monocytes were microscopically
Fig. 3. (A). Expression of viral proteins in long-term surviving PRV-infected monocytes. Monocytes (n=106), at 1 h p.i. with
PRV, were incubated with 0?4 mg PRV-specific polyclonal IgG antibodies ml”1. From 72 h p.i. onwards, expression of viral
proteins in surviving PRV-infected monocytes was determined at different time-points, as described in the text. The total
number (6104) of surviving monocytes is shown (%). The number of surviving monocytes with microscopically detectable
viral antigen expression (#) decreased gradually, whereas the number of surviving monocytes without microscopically
detectable viral antigen expression (n) increased over time. Data represent the means ±SD of triplicate assays. (B) Confocal
sections through the middle of triple-stained monocytes at 72 (left and middle columns) or 144 (right column) h p.i. with PRV,
in the continuous presence of PRV-specific antibodies. Cells were stained with Hoechst 33342 (blue, stains all nuclei), EMA
(red, stains nuclei of dead cells) and FITC-labelled PRV-specific antibodies (green, viral antigens), as described in the text.
Bar, 5 mm.
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H. W. Favoreel and others
positive for viral antigens at 72 h p.i., whereas by 194 h p.i.
less than 20 % of surviving cells showed visually detectable
viral antigen expression. Fig. 3(B) shows confocal images of
monocytes, at different times p.i. with PRV, and cultivated
in the continuous presence of PRV-specific polyclonal IgG
antibodies. Fig. 3(B, left column) shows a PRV-infected
monocyte, at 72 h p.i., that shows expression of viral
antigens and is still alive (positive on Hoechst 33342 and
anti-PRV-FITC but negative on EMA). Fig. 3(B, middle
column) shows a PRV-infected monocyte, at 72 h p.i., that
shows viral antigen expression and is dead (positive on all
fluorescence channels). Fig. 3(B, right column) shows
another PRV-infected monocyte, at 144 h p.i., that shows
no microscopically detectable viral antigen expression
and is alive (only positive on Hoechst 33342). The results
from these experiments indicate that the presence of PRVspecific antibodies suppresses the expression of
PRV proteins in the majority of surviving PRV-infected
monocytes.
The suppressed expression of viral antigens and extended
cell survival induced by PRV-specific antibodies may
perhaps aid PRV to persist in monocytes, circulate in
the blood of vaccinated animals and reach internal
organs. We showed recently that PRV-infected monocytes
with internalized antibody–antigen complexes are able to
transmit virus to vascular endothelial cells via direct cellto-cell spread, in the presence of neutralizing antibodies,
as a possible first step for the virus to reach internal
organs (Van de Walle et al., 2003b). We determined
whether the long-term surviving PRV-infected monocytes
with suppressed expression of viral antigens we describe
here are still able to transmit infectious virus to vascular
endothelial via direct cell-to-cell spread by performing
a co-cultivation assay similar to the one we described
earlier (Van de Walle et al., 2003b). At 144 h p.i, surviving monocytes were collected and co-cultivated with
porcine primary endothelial cells in the presence of PRVneutralizing antibodies. After 30 h of co-cultivation, cells
were methanol-fixed and stained for viral antigens, but no
plaques of endothelial cells could be detected (data not
shown). This indicates that PRV-infected monocytes
with antibody-induced suppression of viral antigens are
no longer able to transmit infectious virus via direct cellto-cell spread. Relating this to the in vivo situation would
mean that antibody-induced suppression of virus may
perhaps help PRV to persist in monocytes, thereby allowing
it to circulate in the blood of vaccinated pigs, but that
external stimuli seem to be needed to allow the monocytes
to resume expression and allow further transmission of
the virus. We are currently examining which factors can
stimulate such resumption of viral antigen expression and
infectious virus production.
Current data suggest that the presence of PRV-specific
antibodies may lead to a suppressed virus infection in a
subpopulation of PRV-infected monocytes. Suppression of
intracellular virus gene expression upon binding of specific
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antibodies to infected cells has been reported already for the
closely related alphaherpesvirus herpes simplex virus type 1
(HSV-1) as well as for the alphavirus Sindbis virus and
has been shown to extend the lifespan of Sindbis virusinfected cells (Chanas et al., 1982; Oakes & Lausch, 1984;
Levine et al., 1991; Griffin et al., 1997, 2001). Although it is
still largely unclear how virus-specific antibodies may
suppress viral protein expression, for Sindbis virus it has
been suggested that transmembrane signalling, triggered by
the antibody-induced cross-linking of viral cell surface
proteins, may be a crucial event (Ubol et al., 1995). In this
context, it is intriguing that especially antibodies directed
against HSV-1 proteins gB and gE (Oakes & Lausch, 1984)
and, as suggested by our current results, against PRV protein
gB, are able to suppress HSV-1 and PRV infection, since
we and others showed recently that antibody-induced
cross-linking of PRV gB and PRV and HSV gE may activate
intracellular signal transduction pathways (Favoreel et al.,
1999a, 2002; Rizvi & Raghavan, 2003). One hypothetical
explanation of how such virus-mediated, antibody-induced
signal transduction pathways can suppress virus infections may be found when considering the mechanisms
of cytokine-mediated suppression of a virus infection.
Binding of several cytokines (especially interferons) to their
respective receptors on virus-infected cells has also been
shown to activate intracellular signal transduction pathways, which, in turn, induce the expression of several
cellular proteins that can interfere with different steps of
the virus life cycle (reviewed by Guidotti & Chisari, 2000).
Perhaps, signal transduction pathways activated by antibodyinduced cross-linking of viral proteins on the cell surface
may induce similar cellular responses. Alternatively, it is
possible that the internalized virus-specific antibodies may
have direct intracellular effects on virus replication. Further
research will certainly be necessary to clarify this issue.
Combining the results from this study indicates that
processes induced by the interaction between antibodies
and viral cell surface proteins in PRV-infected monocytes
may not only delay destruction of the cells by the immunity
but may also enable the cells to survive a PRV infection for
unusually long periods of time by suppressing the ongoing
infection. In turn, this may aid PRV-infected monocytes to
persist in vaccinated animals.
ACKNOWLEDGEMENTS
We would like to thank Fernand De Backer, Chantal Vanmaercke
and Chris Bracke for their technical assistance, and Kevin Braeckmans
for his excellent help with the confocal microscope. This work was
supported by a co-operative research action fund of the Research
Council of Ghent University, Belgium.
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