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Antiidiotypic antibody to reovirus binds to neurons and protects from viral infection.

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Antudiotypic Antibody
to Reovirus Binds to N.eurons and Protects
from Viral Infection
0 .
1 .
Marc A. Dichter, MD, PhD,* Howard L. Weiner, MD,t Bernard N. Fields, MD,$ Gail Mitchell,’
John Noseworthy, MD,§ Glen Gaulton, PhD,§ and Mark Greene, MD, PhD§
A syngeneic monoclonal antiidiotype directed against the idiotype of an antireovirus type 3 hemagglutinin demonstrates several of the biological actions of the original viral hemagglutinin and binds to rat and murine cortical neurons
grown in dissociated cell culture. Receptor-bearing neurons appear within 24 hours of plating in cultures from mouse
or rat cortex taken on embryonic day 15; these neurons are demonstrable for the duration of the culture life span (4 to
8 weeks). When cortical cultures are incubated with antiidiotype before or during exposure to reovirus, the antiidiotype protects neurons from type 3 infection without inhibiting infection of nonneuronal cells with either type 3 or
type 1. Thus an antibody directed against a viral receptor can prevent infection of receptor-bearing cells without
directly neutralizing the virus.
Dichter MA, Weiner HL, Fields BN, Mitchell G, Noseworthy J, Gaulton G, Greene MI:
Antiidiotypic antibody to reovirus binds to neurons and protects from viral infection.
Ann Neurol 19:555-558, 1986
The reoviruses are a family of double-stranded RNA
viruses with three distinct serotypes (Tl, T2, and T3)
that are biologically quite diverse. Two of these serotypes (T1 and T3) have predilections for different parts
of the developing rodent central nervous system
(CNS). Reovirus T1 selectively infects ependymal cells
in neonatal mice and produces hydrocephalus [lo].
Reovirus T3 infects neurons within the CNS, producing an encephalitis in neonatal rodents 1111. The
specific neuronal tropism of the reovirus T3 has also
been demonstrated in vitro using dissociated cell cultures of embryonic rat cortex 157. The tropisms demonstrated by reoviruses T1 and T3 are conveyed by a
single minor surface-coat protein, the sigma 1 protein
or viral hemagglutinin (HA) 117-191. The HA is also
responsible for many interactions between the virus
and the immune system 171. A panel of monoclonal
antibodies directed at the reovirus T3 H A has been
developed and we have identified three epitopes on
the HA, one of which is responsible for neutralization
11). Using cells producing antibodies against the neutralizing site (G5), we have isolated a syngeneic monoclonal antiidiotypic antibody (87.92.6) 1131. This antiidiotypic antibody resembles the reovirus T3 HA in its
binding specificity. Thus its binding site has been des-
ignated Id3 (idiotype 3). The antiidiotype binds to lymphoid cells and cell lines that are known to carry receptors of reovirus T3 and does not bind to cells without
reovirus T3 receptors. It also mimics reovirus T3 in
immunological paradigms, such as the activation of
antigen-specific delayed hypersensitivity and cellmediated cytotoxicity. Most recently we have isolated and characterized the receptor to which this antiidiotypic antibody binds 121 and have shown that it
bears remarkable structural similarities to the betaadrenergic receptor 131.
The antiidiotype can block viral binding to transformed lymphoid cells, although it does not block infection 197. In this paper we describe the characteristics of the antiidiotype binding to rat and mouse
cortical neurons in dissociated cell culture. In addition,
we demonstrate that the monoclonal antiidiotype protects CNS neurons from reovirus T3 infection by
blocking cellular receptors.
From the *Departments of Neurology and Neuroscience, Children’s
Hospital and Beth Israel Hospital, Boston, MA 02115, the “Center
for Neurologic Diseases, Brigham and Women’s Hospital, Boston,
MA 02115, the $Department of Microbiology and Molecular Genetics, Children’s Hospital, Boston, MA 02115, and the §Department of Pathology, Haward Medical School, Boston, MA 02115
Received June 17, 1985, and in revised form Sept 30. Accepted for
publication Oct 1, 1785.
Methods
Embryonic day 15 rat and mouse cortices were dissected and
dissociated in trypsin as reported previously [4]. The cells
were plated onto coverslips coated with collagen and polylysine and were grown in minimal essential medium supple-
Address reprint requests to Dr Weiner.
555
mented with 100 mgldl of glucose, 5% rat serum, 20 pghl
of penicillin, and 20 pglml of streptomycin. Neurons were
identified by their distinctive morphologies under phasecontrast microscopy [43 and by staining with tetanus toxin
and fluorescein isothiocyanate [FITC] antitoxin [ 141.
Cultures were infected by incubation with reoviruses T1
and T 3 (50 pI of 106plaque-forming unitdm1 in physiological saline IPS]) for 2 hours at 37”C, after which 1.3 ml of
complete media was added. The cultures were incubated for
24 hours, fixed in 3.5% formaldehyde followed by cold
acetone, and examined by immunofluorescence, as previously reported {5]. Infected neurons were recognized relatively easily as highly fluorescent cells with distinctive size,
shape, location on top of the background glial layer, and
extensive branching dendritic architecture [5]. In order to
test for antiidiotype protection against viral infection, cultures were preincubated for l hour in antiidiotype (1: 10
dilution in PS) and then washed; the medium was then replaced by virus-containing medium. In some experiments,
the virus was also premixed in media containing the same
concentration of antiidiotype; in others, the cells were not
pretreated with antibody but were infected with virus in the
presence of antibody.
For immunofluorescence with 87.92.6, coverslip cultures
were washed in PS, fixed in 3.596 formaldehyde for 10 minutes, again washed in PS, and incubated for 30 minutes at
room temperature with antiidiotype (ammonium sulfate cut
[ 18 to 55 mdml protein] of ascites, diluted 1:10 in PS). The
cultures were washed three times and incubated with brainadsorbed FITC-goat antimouse gamma globulin (Gibco,
Grand Island, NY) at 1:20 in PS. Coverslips were washed
again, mounted in glycerol, and examined with a Zeiss Universal microscope. Controls consisted of cultures treated
only with second antibody and cultures treated with a variety
of other monoclonal antibodies of the same class.
For double labeling with 87.92.6 and tetanus, cultures
556 Annals of Neurology
Vol 19 No 6 June 1986
were fixed as above, incubated with tetanus toxin (100
pg/ml) for 30 minutes, washed, incubated with unlabeled
goat antitetanus, washed again, and finally incubated with
rhodaminated rabbit antigoat gamma globulin (Gibco, 1:20
in PS). They were then incubated with the monoclonal antiidiotype, washed three times, and incubated with brainadsorbed FITC-goat antimouse gamma globulin, washed
again, mounted, and examined. This technique provided cultures in which all neurons had yellow-red fluorescent stippling around their peripheries, and a subset of these neurons
had green fluorescent somata and proximal processes.
Results
At 24 hours in culture, most of the cells from 15-dayold fetal cortices are neuronal precursors or already
differentiating neurons. More than 90% of the cells at
this time stain with 87.92.6 (Fig 1, A2). By three to
four weeks in vitro, when the cultures are more “mature,” they contain neurons, astrocytes, ependymal
cells, and other nonneuronal cell types of unknown
origin (mesenchymal cells, meningeal cells, and fibroFig 1. Antiidiotype staining of neurons i n young and mature
rat cortical cultures. (Al,2) Same microscopic field from 24-hour
culture photographed in phase contrast (1) and fluorescence (21,
illustrating neurons (arrow) or neuronal precursors stained with
87.92.6. (B1,2j Similar field in culture stained only with
FITC goat antimouse gamma globulin shwing low level of background fluorescence in overexposed photomicrograph. (C) Three
week-old culture, (1) phase contrast, (2) irnmunofluorescencef o r
tetanus toxin, and (3) immunoji’uorescencefor 87.92.6. Note
that on& neurons (arrows) stain for antiidiotype while background cells are negative. All antibodies usedfor zmmunofluorescence and virological studies werefrom hybridoma supernatants
or from ammonium sulfate cuts of ascites {6).
blasts). At this time, only the neurons are stained with
87.92.6 (Fig 1, C3). These cells can be identified as
neurons by their phase-contrast morphologies (polygonal cell bodies with two or more tapering and
branching dendritic processes) or can be labeled with
tetanus toxin (Fig 1, C2), a relatively (but not absolutely) specific marker for neurons in CNS cultures
{14, 153. More than 90% of neurons are labeled with
87.92.6 (Fig 1, C3). The antibody stained the cell
somata and proximal portions of dendrites, but often
spared the more distal processes. No nonneuronal
cells were labeled.
In order to determine if the antiidiotype could interfere with the virus-neuron interaction, 3-week-old
cortical cultures were infected with reovirus T 3 either
after preincubation with 87.92.6 or in the presence of
the antibody, or both. In order to quantitate neuronal
infection, the number of neurons that demonstrated
reovirus immunofluorescence were counted in two
perpendicular scans of each coverslip under x40 oilimmersion objectives. These scans covered approximately 6.4% of each culture and were representative
of the number of infected neurons per individual
coverslip culture. This technique was used to assay for
the effect of the monoclonal antiidiotype instead of
titering the whole cultures because cultures of this age
contain many more nonneuronal cells than neurons
(about 10 to 1) and the antibody did not appear to
affect the degree of infection of the nonneuronal cells.
Thus even a large drop in infected neurons would have
resulted in only a small (or even undetectable) change
in the total viral titers.
In control cortical cultures, an average of 1,100
neurons per 12-mm coverslip culture demonstrated
strong immunofluorescence for reovirus 24 hours after
reovirus infection (Fig 2). When cultures were pretreated with 87.92.6, infected neurons were reduced
by approximately 75%, even if the cultures were
washed prior to incubation with virus. If the virus was
mixed with antiidiotype prior to infection of the cultures, but cultures were not pretreated with antiidiotype, the reduction in neuronal infection was less
pronounced but still present (about 40%). To control
for the possibility that antiidiotype interfered with our
immunofluorescence assay and not directly with infectivity, we infected the cultures with reovirus T3 in the
normal manner (without antiidiotype pretreatment or
presence during the infection) and then, at 24 hours
postinfection, we added the antiidiotype to all of the
antibody incubations during the immunofluorescence
procedure. There was no significant change in the
number of infected neurons stained with the antireovirus antibodies by this procedure.
The infection of nonneuronal cells (astrocytes and
others) in the cortical cultures by reovirus T 3 did not
appear to be affected by preincubation with 87.92.6.
T
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Fig 2. Protection of neurons by antiidlotype. A graph of the
number of neurons per coverslip culture (mean SEMi showing
viral immunojuorescence in reovirus T.3-infected cultures under
control conditions (open bars [ C ] )and after pretreatment of cultures with antibody (hatched bars [Ab]). The pair of bars on
the left show immunoji’uorescence (IF) for the antiidiotype, the
pair on the right for anti-Thy 1 antibody.
*
The antiidiotype also did not block the infection of
nonneuronal cells by reovirus T1. (Reovirus T1 does
not infect neurons in the cultures [5].) Thus 87.92.6
did not act as a “neutralizing” antibody to reovirus T3,
but rather appeared to reduce infection by blocking
neuronal receptors.
Other monoclonal antibodies did not interfere with
reovirus T3 infection of neurons. Figure 2B illustrates
an experiment with a monoclonal anti-thy 1.2 (IgM)
performed identically to that with the 87.92.6. No
significant reduction in neuronal infection was noted.
Similarly, a monoclonal anti-thy 1.1, which stains a
subset of the rat cortical neurons in culture, also did
not block the reovirus T 3 infection of neurons. Thus it
appears that the ability of 87.92.6 to block neuronal
infection by reovirus T3 is specific for both the virus
and the cell type that bears the Id3 receptor (the cortical neuron).
Discussion
Our results show that CNS neurons grown in cell
culture exhibit immunofluorescence staining with
87.92.6, a specific antiidiotypic antibody that was developed against a neutralizing monoclonal antibody for
reovirus T3. The antiidiotype does not stain nonneuronal cells in the same cultures. In addition to identifying receptor-bearing cells, the antiidiotype also has
the capacity to protect neurons from infection with
reovirus T3. It does not protect nonneuronal cells
from either T 3 or type 1 infection. Thus the anti-
Dichter et al: Antibody to Reovirus Receptor
557
idiotype does not have a nonspecific neutralizing action and does not define a universally present, unique
pathway for entry of virus into cells, but appears to
protect by preventing attachment of the virus to the
neurons that bear specific receptors. Recent studies
have suggested that there are structural similarities between the beta-adrenergic receptor and the T3 receptor f3]. Whether reovirus T3 enters neurons via this
pathway is currently being investigated. The mechanism by which nonneuronal cells take up reovirus T3
and support replication is not known, but the lack of
staining or protection by 87.92.6 suggests either that
the uptake is not receptor mediated or that it is
mediated by a receptor with different binding characteristics.
Although 87.92.6 stains almost all the neurons in
either the young or older cultures, only a fraction of
the neurons in the cultures exhibit reovirus immunofluorescence at 24 hours postinfection or longer
IS]. Despite many attempts to change the methods of
infection, multiplicity, age of the cultures, or time after
infection at which we assayed for infected neurons, we
have not been able to induce the majority of neurons
to exhibit substantial reovirus immunofluorescence.
Either the conditions under which the cultures are infected prevent full expression of the viral infection
(i.e., many neurons are “infected” with reovirus but
significant replication does not occur in the majority of
neurons), or many neurons bear T 3 receptors but do
not support viral infection. The former hypothesis
might be tested by in vitro hybridization using T 3
probes, which would detect small amounts of T3 ribonucleic acid rather than requiring the large amounts
of viral proteins for immunofluorescence. The latter
hypothesis would indicate that more than attachment
of the T3 to the neuron is involved in determining susceptibility of neurons to infection E7, 81. This
latter hypothesis is consistent with what is known
about the loss of susceptibility of mice to reovirus encephalitis as they mature, in that reovirus “receptors”
appear to remain present in the brain, even when
encephalitis can no longer be produced [16]. Moreover, other viral components besides the sigma- l
protein can play important roles in neurovirulence
E71.
Antiidiotypic antibodies to viruses have been proposed as potentially useful vaccines to stimulate immune responses to the original virus [8], and may
someday become useful adjuncts to conventional immunotherapy by this mode. To our knowledge, this
report is the first example of an antiidiotypic antibody
to a cell-surface receptor providing direct protection of
the cell from viral infection, although Minor [12} has
reported a monoclonal antibody that blocks cellular
receptors for poliovirus generated by making monoclonal antibodies against the entire surface of the cell.
558 Annals of Neurology Vol 19 No 6 June 1986
Blocking specific viral receptors could become a novel
means of antiviral therapy.
References
1. Burstin S, Sprigs D, Fields B: Evidence for functional domains
on the reovirus type 3 hemagglutinin. Virology 117:146-155,
1982
2. Co M, Gaulton G, Fields M, Greene M: lsolation and biochemical characterization of the mammalian reovirus type 3 cell surface receptor. Proc Natl Acad Sci USA 82:1494-1498, 1985
3. Co M, Gaulton G , Tominaga A, et al: Structural similarities
between the mammalian beta-adrenergic and reovirus type 3
receptors. Proc Natl Acad Sci USA 82:5315-5318, 1985
4. Dichter M: Rat cortical neurons in cell culture: culture methods,
cell morphology, electrophysiology and synapse formation.
Brain Res 149:279-293, 1978
5. Dichter M, Weiner H : Infection of primary neuronal cell cultures with reovirus mimics in vivo patterns of neurotropism.
Ann Neurol 16:603-610, 1984
6. Dimpfell W, Neale JH, Habermann E, et al: ‘251-labelledtetanus toxin as a neuronal marker in tissue cultures derived from
embryonic CNS. Naunyn Schmiedebergs Arch Exp Pathol
Pharmakol290:329-333, 1975
7. Fields B: Molecular basis of reovirus virulence. Arch Virol
71:95-107, 1981
8. Fields B, Greene M: Genetic and molecular mechanisms of viral
pathogenesis: implications for prevention and treatment. Nature
300:19-23, 1982
9. Kauffman R, Noseworthy JH, Nepom JT, et al: Cell receptors
for the mammalian reovirus. 11. Monoclonal antiidiotype antibody blocks viral binding to cells. J Immunol 131:2539-2541,
1983
10. Kilham L, Margolis G: Hydrocephalus in hamsters, ferrets, rats
and mice following inoculations with reovirus type 1. Lab Invest
21:189-198, 1969
11. Margolis G , Kilham L, Gonatos N: Reovirus type 3 encephalitis:
observations of virus-cell interactions in neural tissues. Lab Invest 24:101-109, 1971
12. Minor P, Pipkin PA, Hockley D, et al: Monoclonal antibodies
which block cellular receptors of poliovirus. Virus Res 1:203212, 1984
13. Noseworthy J, Fields BN, Dichter MA, et al: Cell receptors for
the mammalian reovirus: I. Syngeneic monoclonal antiidiotype
antibody identifies a cell surface receptor for reovirus. J Immunol 131:2533-2538, 1983
14. Raff M, Fields KL, Hakomori SI, et al: Cell type specific markers for distinguishing and studying neurons and the major
classes of glial cells in culture. Brain Res 174:283-308,
1979
15. Raff M, Abney ER, Cohen J, et al: Two types of astrocytes in
cultures of developing rat white matter: differences in morphology,surface gangliosides, and growth characteristics.J Neurosci
311289-1300, 1983
16. Tardieu M, Powers L, Weiner H: Age dependent susceptibility
to reovirus type 3 encephalitis: role of viral and host factors.
Ann Neurol 13:602-607, 1983
17. Weiner H , Drayna D, Averill D, Fields B: Molecular basis of
reovirus virulence: the role of the S1 gene. Proc Natl Acad Sci
USA 74:5744-5748, 1977
18. Weiner H , Fields B: Neutralization of reovirus: the gene responsible for the neutralization antigen. J Exp Med 146:13051310, 1977
19. Weiner H , Power L, Fields B: Absolute linkage of virulence and
central nervous system cell tropism of reovirus to viral hemagglutinin. J Infect Dis 141:609-616, 1980
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