Download Immune Serum Increases Arenavirus Replication in Monocytes

J. gen. Virol. (1988), 69, 1735-1739. Printedin Great Britain
1735
Key words: LFV/PV/arenavirus
Immune Serum Increases Arenavirus Replication in Monocytes
By R I C H A R D M. L E W I S , * T H O M A S M. C O S G R I F F ,
B E V E R L Y Y. G R I F F I N , J O A N R H O D E R I C K AND P E T E R B. J A H R L I N G
Medical Division and Disease Assessment Division, United States Army Medical Research
Institute o f Infectious Diseases, Fort Derrick, Frederick, Maryland 21701, U.S.A.
(Accepted 20 April 1988)
SUMMARY
The U937 monocytic cell line was used to determine whether antibodies could
facilitate infection and replication of the arenaviruses, Pichinde virus (PV) and Lassa
fever virus (LFV). When high dilutions of PV-immune serum were added to cultures
simultaneously with PV inoculum, virus replication was dramatically (1000-fold)
increased. Low dilutions of this antiserum neutralized the virus. LFV also replicated in
U937 cells. The presence of LFV-specific immune serum in the growth medium
increased the viral titre as much as 10000-fold. Addition of heat-aggregated IgG
partially inhibited antibody-mediated enhancement, probably by inhibiting the
binding of immune complexes to the monocytic cells.
The arenaviruses Junin virus, Machupo virus and Lassa fever virus (LFV) are major human
pathogens in South America and Africa and cause diseases characterized by haemorrhagic
phenomena and high rates of mortality (White, 1972; Walker et al., 1982; Peters, 1984). The
ability of immune serum to enhance the infectivity of many viruses has been welt established
(Porterfield, 1986); however, this phenomenon has not been studied for arenavirus infection.
Antibody-induced enhancement of viral disease was initially proposed in dengue virus
infection. Halstead et al. (1973) suggested that cross-reacting antibodies might predispose
individuals to the more severe forms of infection, dengue haemorrhagic fever and dengue shock
syndrome. In vitro studies of this disease showed that the target cell of antibody-enhanced
dengue proliferation was the mononuclear phagocyte (Halstead & O'Rourke, 1977a, b).
Enhanced viral infectivity for mononuclear cells in the presence of specific antibody has also
been demonstrated for other viruses (Porterfield, 1986; Cardosa et al., 1986; King et al., 1984;
Burstin et al., 1983; Peiris et al., 1982; Peiris & Porterfield, 1979). Also the presence of immune
serum can increase viral infectivity in monocytic cell lines (Peiris & Porterfield, 1979; Brandt et
al., 1982; Peiris et al., 1982; Burstin et al., 1983; Hotta et al., 1984; King et al., 1984). The cell
lines employed have been well characterized, including demonstration of Fc receptors (Peiris &
Porterfield, 1979; Snyderman et al., 1977; Unkeless & Eisen, 1975; Schlesinger & Brandriss,
1981).
Monocytic cells are central in the immune response to viral infection (Mims, 1986; Morahan
et al., 1985 ; Mogensen, 1979) and possess important haemostatic functions (Edwards & Rickles,
1980; Levy et al., 1981; Helin, 1986). Viral interaction with monocytes might therefore be
important in the pathogenesis of some of the viral haemorrhagic fevers. We have determined
whether immune serum affects arenavirus infection, using the U937 cell line, the arenaviruses
Pichinde virus (PV) and LFV and specific monkey immune serum.
U937 cells were grown in RPMI medium (Gibco) supplemented with 10~ heat-inactivated
foetal calf serum (Gibco) and 1~ each of sodium pyruvate (M.A. Bioproducts, Hagerstown,
Md., U.S.A.), non-essential amino acids (Gibco), and antibiotics. The cells were cultured at
37 °C in humidified air containing 5 ~ CO2, with serial passage twice weekly. The strain of PV
used in these studies was the An 4763 strain originally isolated from its natural host, Oryzomys
0000-8120 © 1988 SGM
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Table 1. Effect of antiserum on virus titre
Experiment 1
M.o.i.
1
O.l
Anti-PV
serum*
loglo P.f.u.t
J'
72 h
c
48 b~
-
0 (0)
+
2.72 (0.09)
-
0 (0)
0 (0)
+
1.03 (0"21)
1"33 (0"39)
96 h
0.8 (1.13)
5.8 (0)
0 (0)
4.47 (0.47)
1.23 (0)
4.4 (0.16)
log10 P.f.u.1Experiment 2
M.o.i.
1
Heat-aggregated
IgG
+
+
Anti-PV
serum*
+
+
-
r
48 h:~
1.52 (1.09)
2.97 (0.34)
1-30 (0.92)
o.oo (o)
A
96 h
3.20 (0.65)
4.07 (0.09)
1.13 (1.60)
3.07 (0-12)
* 1:5000 dilution.
1"Values represent average of triplicate experiments; standard deviation is shown in parentheses.
Incubation time.
albigularis, passaged once through hamster brain and then repeatedly through strain 13 guineapig spleens (Jahrling et al., 1981). LFV was the reference Josiah strain initially isolated from a
patient and subsequently passaged four times in Vero cells. PV antiserum was obtained from a
rhesus m o n k e y inoculated with virus. The monkey demonstrated no illness but developed an
80 % plaque reduction neutralization titre (PRN80) of 1:128. LFV antiserum was obtained from
a rhesus monkey inoculated with LFV. This monkey recovered from a Lassa fever-like illness
and developed a PRN80 titre of 1:200. Both antisera were used at a final dilution of 1:5000.
For infectivity studies U937 cells were washed and pelleted in 50 ml conical tubes at 1600 g for
15 min at 23 °C. Antiserum (or normal monkey serum) and virus were diluted in cell medium,
and 100 ~tl of each was added to the cell pellet. Cells, virus and antiserum were incubated at 37 °C
for 60 min, washed twice in medium and resuspended in 1.5 ml at a cell concentration of 5 ×
105/ml before addition to individual wells of six-well tissue culture plates. Antiserum or normal
serum was again added to a final dilution of 1 : 5000. Cultures were maintained at 37 °C in air
containing 5 % CO2. Samples were removed at various times and infectious virus was measured
as p.f.u, on Vero cell monolayers. Samples for assay were serially diluted and added to cells, after
which virus was allowed to adsorb for 60 min at 37 °C. Monolayers were overlaid with 1%
agarose containing Eagle's basal medium supplemented with Earle's salts and H E P E S buffer.
They were incubated at 37 °C in humidified air containing 5% CO2 for 5 days (4 days when
assaying for LFV p.f.u.). At this time, 2 ml of Puck's saline A containing 1:6000 neutral red was
added to the cultures. Plaques were counted after an additional incubation of 18 to 24 h. PRN80
titres were determined by both the constant serum-virus dilution method and the serum
dilution, plaque reduction method (Jahrling, 1983).
To determine whether immune serum would increase the production of PV in U937 cells, PVimmune serum and two concentrations of virus inoculum were added to U937 cells and the
cultures were incubated and then washed to remove residual virus. The cells were resuspended
in medium containing antiserum and samples were removed for virus titration after incubation
at 48, 72 and 96 h (experiment 1, Table 1). After 48 h, cultures inoculated with an m.o.i, of 1 to
which normal serum had been added contained no infectious virus while antiserum-treated
cultures had supernatant titres greater than 102"5. The increase in virus titre in the presence of
specific antiserum was noted at multiplicities of 1 and 0.1 and at all sampling times.
To test the possibility that increased virus production was mediated by the binding of viral
immune complexes to cellular Fc receptors, heat-aggregated IgG was used to block immune
complex binding (experiment 2, Table 1). Cultures containing antiserum again showed
significantly higher virus titres but addition of the IgG reduced virus titres when measured at 48
and 96 h.
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I
I
I
a
I
~
1737
d,
"= 2
0:_~
10
I
t
100
1000
Reciprocal of antiserum dilution
I
I
10000
Fig. 1. U937 cells were infected with PV in the presence of antiserum. The virus titres are shown in
comparisonwith the dilution of antiserum added to the respective cultures. Sampleswere taken at 96 h
p.i. PV antiserum (O); normal serum (A).
Fig. 1 shows that U937 cells were infected in the presence of various dilutions of PV
antiserum. Serum dilutions were added to a constant number of U937 cells and virus. At the 1:10
antiserum dilution, the viral titre was less in those cultures containing normal serum, suggesting
neutralization. At higher dilutions, the virus titre increased and remained at about 104.
The possibility of antibody-enhanced infectivity of LFV was also tested. As shown in Fig. 2,
U937 cells were capable of supporting virus replication. In the presence of antiserum, the
growth of virus was dramatically increased, resulting in titres of virus increased by as much as
10000-fold.
In these studies, monkey antiserum against PV markedly increased viral replication in U937
cells and heat-aggregated IgG inhibited the antibody-induced increase. Other studies of
antibody enhancement in U937 cells have implicated Fc receptors in the mechanism of
enhancement: only Fc receptor-bearing cells showed enhancement (Unkeless et al., 1975);
F(ab')2 did not mediate increases in viral titre, and monoclonal antibody to Fc receptors blocked
the antibody-induced increase (Peiris et al., 1981).
Some enhancement studies have characterized individual sera for both the ability to
neutralize virus at one concentration and to enhance infectivity at another lower concentration
(King et aL, 1984; Hotta et al., 1984; Peiris et al., 1982; Peiris & Porterfield, 1979; Brandt et al.,
1982). Replication of PV in U937 cells was diminished at high antiserum concentrations. At low
antiserum concentrations, virus titres were significantly increased. This effect might have been
the result of dilution of populations of neutralizing antibodies beyond their effectiveness with
unmasking of populations of enhancing antibodies. Alternatively, the concentration of a single
antibody population, with both neutralizing and enhancing properties, might have been the
determining factor. Studies using monoclonal antibodies (MAbs) against West Nile virus
showed that one of three MAbs could both neutralize virus and enhance infectivity (Peiris et al.,
1982).
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Short communication
a)
I
I
I
(b)
I
j
I
0
//
0
I
48
1
72
I
t
1
96
0
Time after infection (h)
1
48
I
72
1
96
Fig. 2. Cultures of U937 cells were infected with LFV at an m.o.i, of 0.1 (a) and 1 (b). Half of the
cultures contained antiserum (It) and the other half contained normal serum (lq).
Studies in rico have corroborated the evidence in vitro for antibody enhancement of virus
replication for a number of viruses. Passive immunization of rhesus monkeys with dengue
antiserum resulted in increased virus titres in transfused animals (Halstead, 1979). Mice injected
simultaneously with MAb and yellow fever virus exhibited a decrease in mean time to death
which was dependent on the virus strain, the particular antibody and its concentration (Barrett
& Gould, 1986). In contrast, studies using antiserum to treat monkeys and guinea-pigs infected
with LFV have shown a beneficial effect, with no antibody-associated increase in viraemia or
decrease in survival time in either species (Jahrling, 1983; Jahrling & Peters, 1984).
The present studies clearly demonstrate antibody-mediated enhancement of virus infection of
U937 cells for two arenaviruses, PV and LFV. These studies tested only a single PV antiserum
and a single LFV antiserum, but comparisons between sera of different titres and differences in
strain specificity might help to define this phenomenon further. As with other viral infections in
which immune enhancement has been demonstrated, the implications for human disease are
uncertain.
We thank Willis Ennis for invaluable suggestions and Molly Shepley-Stone for typing the manuscript.
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