Download Antibody-mediated Enhancement of Rabies Virus Infection in a

J. gen. Virol. (1984), 65, 1091-1093.
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1091
Key words: rabies~antibody-mediated enhancement/RFFIT
Antibody-mediated Enhancement of Rabies Virus Infection in a Mouse
Macrophage Cell Line (P388D1)
By A. A. K I N G , * J. J. S A N D S AND J. S. P O R T E R F I E L D 1
Central Veterinary Laboratory, New Haw, Weybridge, Surrey and 1Sir William Dunn School of
Pathology, University of Oxford, U.K.
(Accepted 22 February 1984)
SUMMARY
The suggestion that antibodies might enhance rabies virus infection of macrophages
through opsonization of immune complexes was tested in vitro by adaptation of the
rapid fluorescent focus inhibition technique for the examination of a macrophage cell
line (P388D1). Some enhancement of rabies virus infection was shown. The
relationship between such enhancement with the 'early death' phenomenon and its
occurrence in vivo is discussed.
The precise way in which rabies vaccine confers protection against disease, and the
pathogenesis of rabies virus infection are not fully understood. There are occasional failures
even when vaccines of established potency have have used. In both monkeys and mice
immunized with rabies vaccine and subsequently challenged with rabies virus, some die sooner
than non-vaccinated controls given the same challenge (Sikes et al., 1971 ; Blancou et al., 1980).
The mechanism underlying this 'early death' phenomenon is obscure, but it appears to be
mediated by antibody, rather than by cell-mediated immune processes (Prabhakar &
Nathanson, 1981). It has been suggested (Porterfield, 1981) that antibodies might enhance
rabies virus infection of macrophages through their opsonization of immune complexes in a
manner analogous to that which has been shown to occur with dengue, West Nile and several
other viruses (Halstead & O'Rourke, 1977; Peiris & Porterfield, 1979, 1981). An example of
antibody-mediated enhancement of viral infection in vivo occurs with feline infectious
peritonitis virus (FIPV). When non-immune kittens received normal or FIPV immune feline
serum intravenously followed 6 h later by intraperitoneal challenge with FIPV, the antibodysensitized animals developed clinical signs more rapidly and died earlier than did control
animals (Weiss & Scott, 1981).
We have studied the interaction between rabies virus strains and cells of the mouse
macrophage cell line P388D1 (Koren et al., 1975), in the presence and absence of a number of
different rabies virus antisera. Rabies virus does not induce lytic plaques in these cells, but we
have been able to adapt the well-established rapid fluorescent focus inhibition test for rabies
virus-neutralizing antibodies (Smith et al., 1973) by substituting P388D1 cells for the BHK21
cells normally used in this assay. Neutralization curves produced by incubating a fixed dose of
rabies virus with serial dilutions of rabies virus antisera revealed a biphasic response; high
serum concentrations neutralized infectivity, but on dilution the number of foci increased above
the level produced by virus controls without antibody, falling again to reach control levels on
further dilution (Fig. 1).
This type of response was also seen when serum from a person vaccinated with human diploid
cell vaccine (HDCV) was tested with five different rabies virus strains (CVS, ERA, PitmanMoore, Flury LEP and HEP 675). No such biphasic response was seen when similar tests were
carried out in BHK21 cells.
The magnitude of enhancement (two- to fourfold) is substantially less than that found in the
same cell line with viruses of the Togaviridae and Bunyaviridae families, where 30- to 100-fold
increases in plaque counts are regularly observed (Peiris & Porterfield, 1981). Table 1 shows the
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50
45
40
35
30
5
25
t.
o
-= 20
k~
15
10
2
3
4
5
6
-log,0 Antiserum dilution
7
Fig. 1. Twenty-five ~tl of a suspension containing 100 TCIDso of Flury HEP 675 rabies virus was mixed
with 25 ~tl of serial 0-5 log,o dilutions of International Standard rabies virus antiserum (initially 10
International Units/ml) in individual wells of a Costar 96-well microtitre plate. After 90 rain incubation
at 35 °C, 25 ~tl of Leibovitz LI5 medium with 3% heat-inactivated (56 °C for 30 min) foetal calf serum
containing 7.5 x 103 cells were added to each well, and the plates were re-incubated for 5.5 h. An
overlay containing the same medium but including carboxymethylcellulose at a final concentration of
0-75% was then added, using 80 lal/well. After incubation at 35 °C for 4 days, the plates were fixed in
25% formalin for 15rain, washed in phosphate-buffered saline, and stained with fluorescein
isothiocyanate-conjugated anti-rabies globulin. Fluorescent foci were counted using a Leitz SM Lux
microscope illuminated by a 50 W mercury vapour lamp and employing filter system H2, BG38 and
GG 455 edge filters and K530 barrier filters. Mean counts are plotted, based on eight replicates per
serum dilution; bars represent standard deviations; - - - , control.
degree of enhancement seen with a variety of different rabies antisera, together with details of
n e u t r a l i z i n g a n t i b o d y titres as m e a s u r e d b y c o n v e n t i o n a l n e u t r a l i z a t i o n assays i n B H K 2 1 cells
or mice.
I n a s e p a r a t e e x p e r i m e n t it w a s s h o w n t h a t C V S v i r u s r e p l i c a t e d in, a n d i n f e c t i v e v i r u s w a s
r e l e a s e d f r o m , t h e P 3 8 8 D 1 cells in t h e p r e s e n c e or a b s e n c e o f a n t i s e r u m . I n t e r e s t i n g l y , m o r e
v i r u s w a s released in t h e p r e s e n c e o f a n t i s e r u m a t a d i l u t i o n o f 10 - 4 t h a n w a s r e l e a s e d in its
absence (data not shown).
A l t h o u g h t h e s e tests w e r e c a r r i e d o u t in vitro w i t h a c o n t i n u o u s line o f m a c r o p h a g e w h i c h m a y
d i f f e r in a n u m b e r o f r e s p e c t s f r o m r e s i d e n t a n d elicited m a c r o p h a g e s , o u r results s u p p o r t t h e
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1093
T a b l e 1. Ability o f various rabies antisera to enhance the titres o f rabies virus strain H E P 675 in
P388D1 cells
Species in
which antiserum
was prepared
Horse
Man
Rabbit
Mouse
Guinea-pig
Vaccine used
CVS
Human diploid cell
Unknown
Duck embryo vaccine
Flury HEP 675
Serum
neutralization
titre*
NT
10- 2 9 (H)
10 2.o (M)
10 -2.8 (M)
10 -2.9 (H)
Dilution
showing peak
enhancement
10 3.5
10 -4
10 -4
10 4
10 -4
Enhancement
ratiot
2.4
4.1
3.4
3.3
2.5
* Determined in BHK21 cells (H) or in mice (M). NT, Not tested.
~"Number of foci at peak serum dilution/number of foci in the absence of antibody.
h y p o t h e s i s t h a t m a c r o p h a g e s are n o t only c a p a b l e o f s u p p o r t i n g the r e p l i c a t i o n o f r a b i e s virus,
b u t m a y give e n h a n c e d yields o f r a b i e s virus in t h e p r e s e n c e o f r a b i e s virus a n t i s e r u m . I f t h i s c a n
o c c u r in vitro, it m a y also o c c u r in vivo. T h u s , t h e i n t e r a c t i o n b e t w e e n virus a n d a n t i v i r a l
a n t i b o d y d o e s n o t always result in the n e u t r a l i z a t i o n o f viral i n f e c t i v i t y , b u t m a y result in
c o n s e q u e n c e s w h i c h are d e t r i m e n t a l to the host.
The authors would like to thank Dr G] S. Turner of the Blood Products Laboratories for the gift of antisera used
in this work.
REFERENCES
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vaccination and challenge with rabies virus. Journal of General Virology 50, 433-435.
HALSTEAD,S. B. & O'ROURKE,E. J. (1977). Dengue viruses and mononuclear phagocytes. I. Infection enhancement
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cultured murine tumor line. Journal of Immunology 114, 894-897.
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macrophage-like cell lines. Nature, London 282, 509-511.
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macrophage origin - a sensitive assay for antiviral antibody. Journal of General Virology 57, 119-125.
PORTERFIEI.D, J. S. (1981). Antibody-mediated enhancement of rabies virus. Nature, London 290, 542.
PRABrIAKAR,B. S. &NATIJANSOt~,N. (1981). Acute rabies death mediated by antibody. Nature, London 290, 590-591.
SIKES, R. K., CLEARY, W. F., KOPROWSKI, H., WIKTOR, T. J. & KAPLAN, M. M. (1971). Effective protection of monkeys
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SMITH, J. S., YAGER, P. A. & BAER, G. M. (1973). A rapid reproducible test for determining rabies neutralising
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(Received 5 September 1983)
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