Microbiol. Immunol., 39(9), 693-702, 1995
Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody, RG719 Yajin
Ni1,
and
Yuichi
Akihiko
1 Department Japan,
•ôNH•ô
of Molecular
and
Received
Tominaga1,
April
4, 1995.
have
tralizingIgM-type antibody
genicity and
to
MAb
paringthe
The
destroyed whose
for
Morimoto1
point of
the
was
amounts
virus
the
the
while
vaccine
glycoprotein,
that
the
RG719.
which
Kyoto •‚
Microbiology, University,
Present
Yokohama,
Faculty
46-26
address: Kanagawa
of
Sakyo-ku,
Research 227,
Kyoto
Mitsubishi-Kasei
region
two
shared
by
of
suggest
249
to raise
to
the
induced the
the
20-mer
was
to 300.
By
amino
acids
the
com (at
Nishigahara by
leucine
G protein
of
produced the
is by
presumed
virus-neutralizing
in humans
ERA
epitope
phenylalanine-263
peptide 268)
anti-
(HEP, the
Nishigahara
that
20-mer
positions also
on
that the
phenylalanine-263
generated
synthetic
mim
epitope
antibodies
and
rabbits
in
production
peptide.
Monoclonal
antibody,
Synthetic
peptide
19,20). In the last decade,the G protein moleculehas exten sivelybeen studied at the molecular level to define its molecularstructuresand the functionaldomainsinvolved in G protein functions as well as the antigenic determi nantsand other domain(s) responsiblefor the neurovirulence of the virus. Studies on the antigenic structures would provideus a lot of useful informationfor, such as, epidemiologicalstudies and developmentof new rabies vaccines. Neutralizingmonoclonalantibodies(MAbs)as well as other modemtechnologies(e.g., the gene cloning Abbreviations: sulfate;
of
ABTS,
BSA,
bovine
FITC, IPTG,
Ltd.,
centfocus
693
2'-azinobis-(3-ethylbenzthiazoline)-
DEAE,
immunosorbent
fluorescent
albumin;
cDNA,
assay;
isothiocyanate;
MAb, inhibition
monoclonal test;
TBS,
complementary
diethylaminoethyl; FA, IgM,
isopropyl-ƒÀ-D-thiogalactopyranoside;
hemocyanin;
Japan.
2, serum
acid;
enzyme-linked
Japan. Co.,
242
not
Sciences, 606-01,
position this
with
Department
Pharmaceutical
Kyoto, Inst.,
Kawai,
strains that
in
observations
epitope,
three
indicated
are
in Japan
reacted
Sequential
Dr . Akihiko
606-01,
virus-neu
tested,
but
was
acid
other
from
replacement
and
produced
the
G protein
deoxyribonucleic to
Kyoto
indicated
strains
found
The
amino RG719
virus
we
Amongfive viral proteins (L, G, N, M1and M2)which composethe maturerabies virion,only G proteinis a gly coproteinand its homo-trimercomposes a spike projec tionprotruding from the virion surface. G protein is essential for initiating the infection of the virus, that is, for viral adsorption and penetration,as well as for prog enyvirus formationby budding at the cell surface. In in vivoinfection,the G protein function(s)is also shown to be responsiblefor displayingthe neurovirulentnature of teh virus (5, 15, 17). In addition, G protein is a major element for inducing the specific immunity against the rabies virus infection, and the virus-neutralizing anti bodiesraised in animals are directed to G protein (3, 11, correspondence
Kyoto,
a rabies analysis
strains
epitope These
MAb from
antibodies
those
produces
rabies
ranging
strains,
of
phenylalanine.
to MAb
HEP
four
revealed
for
of the
of G protein,
to three
(ranging
the
of
while
of
specifically
with
words•ôNS•ô: Rabies
Molecular
Sakamoto2,
University,
Immunoblot
strain,
mutants
structure
epitope
sequence
to bind
Vaccination
*Address
, Shin'ichi
, Kyoto 860, Japan
which
Nishigahara deletion
G protein,
RG719 RG719). four
the
with
line
as MAb
Among
common
by
Sciences Kumamoto
of G protein.
mutagenesis
the
cell to
sequence
HEP
replaced
acid
hybridoma
from
are
Pharmaceutical Kumamoto,
(referred
primary
acid
which
constructing
shown
significant
Key
Kinjiro
1995
Studies
of the
of Inst.,
epitope
absent
amino
epitope
amino was
rabbits.
•ôNH•ô
was MAb.
291)
leucine-263
ickingthe
of
a sequential
site-directed the
essential
region
a murine
region
and
29,
antibody
estimated 263
May
established
to the
in a middle
strain.
Faculty
monoclonal
RG719
reacted
positions
Honda*,
Research
Accepted
recognized
CVS)
located
Microbiology,
2Chemo-Sero-Therapeutic
Abstract•ôNS•ô:We
the
Yoshikazu
Kawai*,1
antibody; Tris-buffered
ELISA,
fluorescent
antibody;
immunoglobulin KLH,
keyhole
RFFIT,
rapid
saline.
M; limpet fluores
694
Y. NI ET AL
and nucleotidesequencing)have facilitatedthose studies. In addition, escape mutants resistant to the neutraliz ingMAbs are helpful for identifying the epitopes on the G protein molecule. Especially, identification of the MAb-specifiedneutralizinglinear epitopes may be a promising approach to the development of synthetic peptide vaccines. Accordingly, it was of interest to have a list of linear epitopes on the G protein. Most of the G protein-specificMAbs reported so far, however, recognizeconformationalepitopes;only one MAb-spec ifiedlinear epitope has been reported (1, 4). In this study, we have tried to establish hybridoma clones which produce MAbs against the linear epitopes on G protein. Among more than twenty G protein-spe cifichybridomaclones,we haveobtainedonly one clone (namedRG719)whichproduceda linearepitope-specific antibody. We investigated the epitope of this antibody. The antigenic site was mappedto a phenylalanine-263containingregion: substitutionof phenylalanine-263by leucine abolished the antigenicity of the HEP G pro tein.We could also detect significant amountsof anti bodiesagainst this epitope in humans who were vacci natedwith the rabies HEP vaccine.
vector
was
a small
made
N-terminal
a whole coli
G
in the
in
presence
of
the
of
inserted
in the
tionmutants of
a synthetic
reconstructed
combinations
of
Nhe
I linker
Fig.
3.
restriction
into
the
Site-directed were
by
using
into
clone by
of
strain
et al (16). site
and
5'-CTG
were
point
same
an
as
illustrated
in
mutations
and
Nishigahara method The
into
TCA
CGA
GAC
TCG
primers
GAT
for
HEP
Introduction
confirmed sites
described
synthetic
AGA
TTT
cDNA
inserted Two
TTA
by
G
one
were
respectively.
mutation
various
inserting
vector.
GAC
was
the
the
mutagenic
G proteins,
of
(12).
phase
the
mutations
am4
various
by
mutation
GAC
as
of
point
HEP
cDNAs
CAC
used
Nishigahara
the
the
5'-CAC
and
GAA-3',
of
was The
of
oligonucleotides, GAG-3'
G gene
M13mp19
Nishigahara
polylinker
sites
double
vector
Sakamoto
the
the
gene dele
with
or
Single
a simultaneous
a phage
out gene
at various
mutagenesis.
introduced
strains
the
G
several
cutting
enzymes,
gene
the
vector,
from
and
isopropyl-ƒÀ-
From
by
sequence
of
Escherichia
inducer,
pUC18
generated
DNA
composed
the ƒÀ-galactosidase
(IPTG).
were
lengths
protein
lacZ-deficient
D-thiogalactopyranoside
by
as
of
nucleotide
described
previous
ly(13). Production
Rabiesviruses. The HEP-Flury,CVS andERA strains havebeen describedpreviously(8, 14). The Nishigahara strain is a standard rabies virus strain which has long been used as a seed strain for producing animal rabies vaccines in Japan (16). Infectivity was assayed by plaque formation (9). Establishmentof hybridomas. Spleencells were iso latedfrom Balb/c mice immunizedwith UV-inactivated purified rabies virions (HEP-Flury strain), fused with myeloma cells (NS-1) by stirring in 50% polyethylene glycol 2000 (6), and then incubated in HAT medium. After screeningby enzyme-linkedimmunosorbentassay (ELISA) using the fixed antigens of rabies virus-infect edcell lysates,aboutone hundredrabies-specifichybrido maclones were isolated,from which more than twentyG protein-specificclones were chosen by FA staining, and confirmed by immunoprecipitation and immunoblot analyses. Among such G protein-specificclones, only one clone (RG719) produced MAb that was capable of recognizing the SDS-denaturedG protein. The isotype of MAbs was determined by double immunodiffusion assays (10) using an isotyping kit (Miles Laboratories Inc.). Preparation of deletion mutants of the G gene. The cDNA clone (pHP452) of HEP G protein (13) was inserted into the polylinker site of the lacZ gene in frame of a plasmid vector pUC18; the reconstructed
a fusion
fragment
protein,
sequencing
Materials and Methods
to induce
mutant of
of
G gene
pUC18,
and
mutant
G
lysates
were
were
proteins
first
subjected
process
to was
were
increased
the
sets
in
Then,
was
stained
using
control)
or
MAb
conjugated
goat
(BA85,
RG719,
and
anti-rabbit
or
was
genes
in were
expression structed sown
vectors on
described fection,cells
previously were
G
to
their
were
sub
were
first
subjected
each
was
then
blotted
&
Schuell),
into
the
blocking
then
for
studies
with
the Ig
color of
by
the
(12).
mutant
site
fixed
with
G
of
(2). into
cold
4-
proteins mutant
G
a retroviral The
recon-
BHK-21
DEAE-dextran 48
peroxidaseantibody.
and
BamHI
After
and
(positive
development.
Wild-type
transfected
to
solution
antiserum
pZIP-NeoSV(X)1 were
coverslips
mutant due
anti-murine
cells.
inserted
most
rabbit
used
BHK-21
vector
products
Schleicher a
anti-G
Immunofluorescence expressed
a rab
A-insoluble
G protein
and
with
the
Chloro-1-naphthol
cell
analysis.
treated
either
of the
with protein
precipitants
Samples
filter
then
gene
Samples
probably the
SDS-PAGE
a nitrocellulose
which
and
buffer.
because
amount
method. 10%
lysis
mutant
necessary,
immunoblot
of
IPTG,
and
the
Each
lacZ
Expression
immunoprecipitation
enrich
small
Immunoblot
IP
coli.
the
coli.
by
antiserum
instability).
jectedto
onto
to
in order
proteins
two
with
E.
into
E.
induced
prepared
in
in frame into
was
anti-G
(Sigma)
G proteins
inserted
tranfected
bitpolyclonal
(this
mutant
was
cells
method to
acetone,
72hr
of and
as trans
stained
A SEQUENTIAL first
with
anti-G
rabbit
and
then
stained
with
bitor
anti-murine
under
a Nikon
ELISA
well
case
peptide
of
filter
(BA85,
well
PVC
the 20%
was
goat sera
then
to
goat
antibody
and
incubated the
for
bound
each
well
line)-sulfate
to
a 96-
plate
(or then of
incubated the or
1hr
peroxi
anti-human
well
of
the
washing
quantified
the
for
Then,
each
of
and
After
was
in
the
dilutions
and
37C.
peroxidase
(ABTS)
placed
anti-rabbit
at
each
BSA/TBS
TBS.
added
the
nitrocellulose
TBS,
fivefold
with
2,
(Falcon).
onto
and
5% or
1hr
mogenicsubstrate,
adsorbed
plate
of
Two-
was
were
adsorbed
with
virions
(G249-268),
with
anti-murine,
IgG
TBS,
was
washed
dase-conjugated
antigen
& Schuell)
added
and
PVC
washing
serum/TBS.
using
plate
Fig.
with
tralizing
the
ELISA
1.
activity
a chro
reaction
kit
A
a set
Bakelite
determined
at
Assay
of
tralizing
420nm
of
solution
the
8-well
for
60min
Color
antibody by
In brief, the
was
titer
Virus-neu-
a slightly
modified
0.1ml
serum
of
and
(104PFU/ml) Lab-teck
intensity
a spectrophotometer.
determined
(18).
dilutions
virus
using
was
method
serial
Tokyo).
virus-neutralizing
titer
RFFIT
Co.,
0.1ml
were
chamber
two-
or of
mixed
in
slide.
the
After
with
well
and for
incubation fixed
and
sample
and
berof
growth
medium
at 37C
for
and
cells
the
fields
to each
the
incubation
slide
cells
suspended
well
and
was
fixed
Numbers
were
at
foci
of
a low
acetone
HEP
with
FA-positive
HEP
strain; •œ,
of
oligopeptides. sized
by
serum
Twenty-mer a solid
acid
sequence
(21).
They
phase of
coupled
rabbits several
G
to to
with
the
the
VSV-NS
keyhole
Lerner's
synthethe
limpet
peptide
were
hemo and
Freund's at
inoc
in
an
RFFIT of
lished,only which
one reacted
than (named with
the
hybridoma
RG719) SDS-denatured
produced
the
dilution was
petri
were
of
dishes.
incubator,
the for
10
the
cov to
15
anti-rabies
counted
N
for
antibody
expressed
each
(the
as
num
100%).
Sym
strain.
clones the G protein
estab
antibody of
The
same
neutralizing
HEP
RG719 G antigen
only
very
weakly
21
cells
infected
with
while
with with with
stained not
stained
the
antibody), Nishigahara almost
other
two
were
anti-rabies
acetone-fixed
the strains
the
rabbit
anti-G
antibody,
the
MAb
(data
the
ERA
and
the
MAb, with
cells
was
antibody
virus-infected
rabbit
at all
performed G antiserum.
infected
Nishigahara the
with
the
the
studies
both
the
the
shown).
a rabbit
in
stained MAb,
stained
were
and
of
a
activ
displayed
against
not
immunofluorescence
or
cells
data
by
neutralized
of
also
activity
CVS;
MAb
the
antibody
Virus-
focus-reducing
infectivity
a
RG719
examined
RG719
protein/ml
the
by
MAb
shown).
was
(50%
1-2ƒÊg
1).
positively the
at
(Fig.
The
we
MAb
strain
strain
using
obtained.
The
HEP
affect
Isotyping that
not
assay.
not
and
(data antibody
obtained
(ERA
assays. showed
the
the
of
it did
Double
inter
RG719
twenty
35-mm
CO2
fields
cultures
antibody
while
were
more
ten
test
activity
infectivity
and
Among
further m.o.i.=
using
immunoblot
IgM-type
positively
of MAb
(anti
were
temperature
Nishigahara
the
modified
adjuvant.
two-week
Results
Characterization
in
at room
immunodiffusion
itywas
amino proteins
method,
strain
neutralizing
synthetic
were
and
(inoculated
against
the
mimicking
complete
boosters
vals),anti-sera
by
virus
according
ulatedinto
against
oligopeptides method
rabies
were
cyanin(KLH)
After
immune
mixtures
in a 5%
in
mixed
solutions
(105cells/0.2ml;
coverslips
foci
against infected
double was
magnification
(e.g., •~160). Production
of
RFFIT
protein/ml).
immunofluorescence
number
plotted
the
an were
in
studies of
counted
on
RG719
stock=3mg
37C,
acetone
by
Virus-neuby
incubated
with
immunofluorescence
antiserum.
10
BHK-21
added
Then, to
anti-N in
105
were
48hr.
subjected
rabbit
37C,
at
at 37C
with
untreated
MAb
suspensions
plated
RG719. assayed
(103PFU/0.1ml)
original
1hr
48hr
examined The
the
MAb
was
diluted
cell
After
bols:•¢,
at
for
BHK-21
antibody.
of
5-fold
0.01PFU/cell),
of
RG719
suspensions
of
incubation
mixed
min,
rabies
each
virus serially
erslipswere
fivefold
MAb
bodyconcentration After
(Sumitomo
of
activity of
Rabies
with
color
Virus-neutralizing
method.
2'-azinobis-(3-ethylbenzthiazo of
695
examined
rabies
(5mm•~5mm)
blocked
were
at 37C,
plastic
100ƒÊl
After
GLYCOPROTEIN
anti-rab
were
protein/well)
Schleicher plate.
filter)
test
in
VIRUS
RG719,
goat
Purified
peptide
pieces
OF RABIES
microscope.
a 96-well
synthetic
square
MAb
They
titer
2.5ƒÊg
of
murine
antibody.
antibody
(10ƒÊg)
small
or
FITC-conjugated
epifluorescence the strain;
each
In the
Ig
for
(HEP-Flury onto
antiserum the
EPITOPE
not CVS
either
but
but
cells not
shown).
at
strains
were
the
uninfected
antibody
all
BHK-
(data
also
not
696
Y. NI ET AL
Fig. 2. Immunoblotanalysisof the rabiesvirusG proteinwithMAbRG719.Purifiedvirionsof four strains (HEP,Nishigahara,ERA and CVS)were appliedto two setsof 10%SDS-PAGE. Aftertheelectrophoresis, viralproteinsin the gelswere blottedontonitrocellulose filter and immunostainedwith rabbitanti-Gpolyclonalantibody(PAb)or MAb RG719as describedin "Materialsand Methods."Left: rabbit anti-GPAb; right: MAbRG719.Virussamples:(fromleft to right)HEP,Nishigahara,ERA, CVS, and control (no virion).
Fig. 3. Preparation of deletion mutants of G protein. Deletion mutants were generated by inserting a universal Nhe I linker into the HEP G-cDNA or deleting a fragment from the cDNA by digestion with appropriate restriction enzymes (see "Materials and Methods"). The name of each mutant correlate to the amino acid positions of the deleted sequence (G wt: wild type G protein). Reactivity of each mutant with anti-G PAb and MAb RG719 is also depicted to the right based on the data shown in Fig. 4.
shown). Immunoblot analysis also gave a similar reactivity profile of the MAb with the four rabies virus strains as seen in the virus-neutralizationand immunofluorescence studies; the MAb did not bind to the G protein of Nishigaharastrain, but did to that of the other three strains(HEP,ERA and CVS;Fig. 2). The reactivity on the immunoblots indicated that MAb RG719 recognized a sequential epitope of the G protein. Consequently,we next triedto map the epitopeon the G protein molecule. Studies with Deletion Mutants of G Protein To roughlymap the epitopeon the G proteinmolecule of HEP virus, several deletion mutants of the G gene were generatedusing the HEP G-cDNAinsertedinto the polylinker site of a plasmid vector pUC18 (Fig. 3; see also "Materials and Methods"). The mutant G proteins were expressed in E. coli, and the lysates of the transformedE. coli were analyzedby an immunoblotmethod.
The samples were first precipitatedwith a rabbit anti-G antiserum to concentrate mutant G proteins, and the immunoprecipitantswere then subjected to immunoblot analysiswith MAb RG719. As shownin Fig. 4, deletion mutantsof G protein,GA1-122,GA123-214,GA400-505 reacted with the MAb, while G0214-505, GA242-300 and GA242-505did not, suggestingthat the epitope was located in a region ranging from amino acid positions 242 to 300 of the G protein. The results are also summarized in Fig. 3. Based on this assumption, we compared the amino acid sequencesin the 242-300 region of the G protein of the four rabies virus strains, and found two candidate amino acids (at position 263 or 291) which may be essential for constructing the epitope (amino acids at positions 263 and 291 of the Nishigahara G protein were substitutedby leucine and isoleucine,respectively; Fig. 5). As to the two candidate amino acids, we assumed that the amino acid substitution at position 263
A SEQUENTIAL
EPITOPE
OF RABIES
VIRUS
GLYCOPROTEIN
697
Fig. 4. Immunoblotanalysisof deletionmutantsof HEP G protein. The mutantG cDNAswere expressedin E. coli by IPTGinduction,and subjectedto the immunoblotanalysis;the lysateswere firstprecipitatedwitha rabbitanti-Gantiserumand proteinA-insoluble (Sigma)to concentratethe proteins,and were thenappliedto two setsof 10%SDS-PAGEfor immunoblotting (see"Materialsand Methods").Antibodies:(top) rabbitanti-G PAb;(bottom)RG719.Wild-typeand mutantG proteinsare indicatedby asterisksto the rightof theirbands(bandsof degradationproductswere notmarked)."H" and "L" shownon the toppanel indicatethe bandsof heavyand light chains of the rabbit anti-G PAbused for G protein concentration.Two mutants,whose bands were concealedby a broad band of immunoglobulinheavychain,were appliedagain to the SDS-PAGEgel withoutconcentrationfor immunoblotting,and shownto the rightof the top panel. was most likely to have affected the antigenicity of the Nishigahara G protein, because the substitution of phenylalanine-263 by leucine seemed to be more influential due to removal of the aromatic structure from the presumed epitope site than the replacement of methionine-291 by isoleucine. Site-Directed Mutagenesis To examine the assumption mentioned above, we introduced a point mutation into the G gene of HEP and Nishigahara strains using a phage vector M13mp19
am4 as described in "Materials and Methods." The point mutations were as follows: phenylalanine-263of HEP G proteinwas replacedby leucine, and leucine-263 of Nishigahara G protein by phenylalanine (Fig. 5). The mutant G genes were transferred into pUC18 -and expressed in E. coli by induction with IPTG, and the products were tested for their reactivity with MAb RG719 by immunoblot. The amino acid substitution at 263 of HEP G protein [GHEP (F263L)] abolished the reactivity with MAb RG719 (Fig. 4), while the Nishigahara G protein [GNis(L263F)] recovered the anti-
698
Y.NI
ET AL
Fig. 5. Comparison of amino acid sequence from positions 241 to 300 of G protein of the four ravies virus strains. The amino acid sequence from positions 241 to 300 of the G protein of four rabies virus strains (HEP, Nishigahara, ERA and CVS) was arranged to be compared each other. Only substituted amino acids were depicted for the latter three strains. An amino acid at position 263 (indicated by an asterisk) was assumed to be the candidate which would have affected the reactivity of Nishigahara G protein with MAb RG719 (see text). Point mutations were introduced at position 263 of G protein of HEP and Nishigahara strains to replace phenylalanine and leucine by leucine [GHEP (F263L)] and phenylalanine [GNis (L263F)], respectively.The synthetic 20-mer peptide, G249-268, was prepared by mimicking the underlined sequence (from positions 249 to 268) of the HEP G protein.
Table 1. Conversion G proteins
of the antigenicity
of HEP and Nishigahara
with MAb RG719 by a single amino acid substitutiona)
a) Single amino acid substitutions were introduced at position 263 (see Fig. 5) using the G cDNA of HEP and Nishigahara viruses as described in "Materials and Methods." Wild-type and mutant G proteins were expressed in. E. coli and BHK-21 cells, and examined for their reactivity with MAb RG719 and rabbit polyclonal anti-G antibody (PAb) by an immunoblot method (see Fig. 4) and immunofluorescence study.
by replacementof leucine-263by phenylalanine (data not shown). These results provide convincing evidencefor the assumptionthat the amino acid at posi tion263 is included in the MAb719-specific epitope and phenylalanine-263is an importantelement to con structthe functional epitope. Point mutants were further checked by immunofluo rescencestudies. The mutant G genes were inserted into the BamHI site of an expression vector, pZIPNeoSV(X)1and expressed transiently in BHK-21 cells (see "Materials and Methods"). The transfected cells were fixed with acetone and doubly stainedwith a rabbit polyclonalanti-G antibodyand MAb RG719to examine the antigenicityof the mutant G proteins. Table 1 shows the results;GHEP (F263L)did not react with MAb RG719, while GNis(L263F) regainedthe antigenicity,giving evi dencefor the assumption that phenylalanine-263is an importantelement of the epitope for MAb RG719.
Experiments with the Epitope-MimickedSyntheticPep tide We next examined whether the linear epitope identi fiedby MAb RG719 is effectivefor raisingantibodiesin animals and humans immunized with the rabies vac cine.For this purpose, we tried to synthesize some 20mer oligopeptidesby mimickingthe aminoacid sequence in the region containing phenylalanine-263 (i.e., from positions249 to 278) of G protein; the designedpeptides were named G249-268, G254-273 and G259-278, respectively, according to the position of the starting and terminatingamino acids on the G protein molecule (Fig. 5). The peptide G249-268 was successfullysyn thesized,but the synthesis of the other two (G254-273 and G259-278)was not successful in spite of our efforts to solve unexpected technical problems encounteredin the solid phase peptide synthesis. Accordingly, we could use only one peptide(G249-268)for the following experiments. The syntheticpeptide was firstchecked for its specific binding to MAb RG719by ELISA(data not shown),and then coupled to KLH and inoculated into rabbits with complete Freund's adjuvant. The antisera against the peptide was obtained four weeks after the third booster. The antibody titer against the peptide was assayed by ELISA (see "Materials and Methods"), and the maxi mumtiter was about 1:600 (data not shown). Similar titers were also obtained by using rabies virions (HEP strain) as the antigen (data not shown). Immunoblot analysis showed that the anti-peptide antiserum dis playeda similar reactivity profile with the virions of the four strains as seen with MAb RG719 (Fig. 6 and Table 2). RFFIT assaysdemonstratedthat the anti-peptideanti serumcontained the virus-neutralizingantibodies(50%-
A SEQUENTIAL
EPITOPE
OF RABIES
VIRUS
GLYCOPROTEIN
699
Fig. 6. Immunoblot analysis of the anti-peptide antiserum. Purified rabies HEP virions were applied to two sets of 10% SDS-PAGE. After the electrophoresis, the virion proteins separated in the gels were blotted onto the nitrocellulose filters for immunostaining using MAb RG719 and the rabbit serum against the synthetic peptide (G249-268), which was obtained four weeks after the third booster, as described in "Materials and Methods." Left: polyclonal anti-G antibody; right: anti-peptide antibody. Virion samples: (from left to right) HEP, Nishigahara, ERA, CVS, and control (no virion). Table 2. Reactivity strains with MAb
" Reactivities samples
profile of the G protein RG719
in Figs.
of various
anti-peptide
of four strains of rabies
were summarized
ses shown
and rabbit
rabies virus
antibody"
virus with three antibody
from the results
of immunoblot
analy-
2 and 6.
neutralizingactivity was observed at a dilution of 1:60; Fig. 7). The neutralizing titer was decreasedwhen the antiserum was preincubated with an immunosorbent (proteinA-insoluble,Sigma) which is known to specifically remove the IgG-type antibodies from the rabbit serum (Fig. 7). In addition, the serum similarly neutralized the infectivity of the ERA and CVS strains, while the infectivity of the Nishigahara strain was not affected by the serum (data not shown). These results strongly suggested that the synthetic peptide G249-268 might well serve as the RG719-specific antigen. Accordingly,we used this peptide againto examine whether RG719-like or linear epitope-specific antibodiescould be induced in rabbits and humans vaccinated with a current rabies vaccine (HEP strain) produced in Japan. Rabbits immunizedthree times with the HEP vaccine at a two-week interval were shownto produce a significant amountof antibodies against the peptide (Fig. 8A). We could also detect the antibodies reacting with the peptide in humans who received the rabies vaccine. Figure8B shows a representativeresult, showing that significantamounts of the peptide-specific antibodies were producedwithin one month in a human immunized once with the vaccine, and the antibody titer was increased after the second vaccination.
Fig.
7.
The
rabbit
Virus-neutralizing
activity
anti-peptide
tralizing
antiserum
antibody
immunosorbent serum
(protein
was
protein
titer
mixed
with
A-insoluble was
incubated
trifuged
in
a refrigerated
ed
and
and
HEP bols: •›, treated
protein
tested
virus
by
A)
on
the
three
suspended
mixture
untreated
and
for RFFIT
untreated
on
of was
the the
effect titer.
a rotator
of
sera
were
anti-peptide
(see
"Materials antiserum; •œ
latter 10%
virus-neuwith
test,
at 4 C, X g
serially
MEM. and for
then 5 min.
twofold
activity and
the
anti-
suspension
Eagle's
10,000
virus-neutralizing
assay
the
overnight at
its
pretreatment
a serum-free
microfuge
their
For
antiserum. for
of
volumes in
A-treated
anti-peptide checked
against
Methods").
of The cenThe dilutthe Sym-
immunosorbent-
antiserum.
Discussion Among more than twenty rabies virus G protein-specific hybridomacloneswe established,only one (RG719) was shownto producea linearepitope-specificMAb, and the others produced conformational epitope-specific ones. We can think that there are many sequential epitopes on the G protein molecule,but most of them would not be so strong as conformationalepitopes. The linear
700
Fig.
Y. NI ET AL
8.
from
Detection
the
endfor
Fig.
PVC
plate.
37C. was
of antibodies
vaccinated 5)
by
After
After
, standard raised
the
as
(A): •¡,
against
, human
the serum
described
one
the
in from
month
was
were
"Materials
from peptide after
Methods," three
(G249-268). the
5-fold
and
second
immunized (B): •¡, vaccination.
rabbits the
to
diluted with
with
small
the
several
vaccinated
which pieces
with
recognize
the
of nitrocellulose
solutions
were
color
times human
intensity
with
the
serum; •›,
determined with
the
to
placed the
Sera
peptide each
and for
420nm
one
vaccine.
in
wells
well
at
leg
a 96-well for The
1hr
at
color
a spectrophotometer.
G protein; •œ, after
the
of
37C.
preimmune
month
obtained
(see
incubated
1hr
with
vaccine; • , whole
serum
HEP 20-mer
antibody at
HEP
SDS-denatured human
rabies
synthetic
second was
interval
the
filter added
peroxidase-conjugated
at a two-week
preimmune
humans
antiserum the
and times
and
antibodies
advance
incubated
immunized a rabbit
in for
in
serially
filters
a rabbit
epitope examined
adsorbed
procedures, TBS,
synthetic
RG719 were
peptide
blocking
antiserum
the
humans
The
with
serum
anti-G
and
ELISA.
washing
developed
Antisera.
against
rabbits
rabbit rabbit
the
first
serum;•› antiserum
vaccination;•œ
A SEQUENTIAL
EPITOPE
OF RABIES VIRUS
epitope-specificMAb, named RG719, displayed virusneutralizing activity against the HEP, ERA and CVS strains similarly, but the infectivity of the Nishigahara virus was not affected at all. Immunoblotand immunofluorescencestudies also demonstratedthe absenceof the antigenicityon the Nishigahara virus G protein. Our finding that the Nishigahara strain lacked the RG719-specificantigenicityhelpedus to performthe epitope mapping. The antigenicsite for MAb RG719could be mappedto a phenylalanine-263-containingregion of the G protein molecule,which was further evidencedby introductionof a single amino acid substitutionat position 263 of G proteinof the HEP and Nishigaharastrains, resulting in the loss and re-acquisition of antigenicity, respectively. The RG719-specificepitopesite was very close to the site recognized by another linear epitope-specificMAb (6-15C4; 4). From the studies with escape mutants, Dietzschold et al (4) demonstrated that the antigenic site for MAb 6-15C4 was destroyed by a single amino acid substitution at position 264 (arginine)by histidine. The epitope for MAb 6-15C4, which was shown to be conserved in almost all rabies virus strains, was further characterizedby Heijdenet al (7) using a set of octapeptides that were synthesized to mimick the core amino acid sequence(L-H-D-F-R-S-D-E)of the presumedantigenic site but to have a single amino acid substitutionin the core sequence of the epitope. From their studies, phenylalanine-263was also shown to be essential for preservingthe epitope for MAb 6-15C4, indicatingthat the epitopes for MAbs RG719 and 6-15C4 strongly resembleeach other. At present,however,we cannottell whether the epitope structures are the same or not. Although phenylalanineat position 263 is conserved in almostall rabiesvirus strains(7), and is very important to construct the epitope for MAbs 6-15C4 (7) and RG719,it does not seem to be essentialfor the G protein to perform its functions. In addition, phenylalanine263 may not endow the virus with any selective advantage, becausethe Nishigaharastrain had lost it probably duringthe serialpassagesthroughlaboratoryanimalsand cell cultures (16). Since the rabies vaccines of Nishigahara virus have been effective to induce protective immunity in animals against the challenge virus infections (16), the RG719-specificepitope does not seem to be indispensablefor the vaccine. This epitope, however, seemed to actuallyinduce the neutralizing antibodies in animals. The rabbit polyclonal antibodies induced by a 20-mer oligopeptides displayedneutralizingactivityagainstthe viral infectivity of the HEP, CVS and ERA strains, but not the Nishigahara strain. The anti-peptide antibodies showed a similar binding pattern as MAb RG719 in immunoblot
GLYCOPROTEIN
701
assays. These results indicate that the peptide covers the whole epitope structure to induce the neutralizing antibodies in animals. By using this peptide, we further detected significant amounts of RG719-specific antibodies in the human bodies vaccinated with the rabies HEP vaccine. The anti-peptide antiserum was also shown to contain IgG-type antibodies as evidenced by removal of the neutralizing activity by preincubation with protein A-insoluble. These results may lead to the development of a peptide vaccine, for which, however, we have to perform more fundamental studies, such as to find more neutralizing linear epitopes on the ,G protein molecule and the ways to use the peptides for vaccination. In this regard, for instance, Dietzschold et al (4) showed that the protective immune response could be induced in mice with the synthetic peptide which was composed of two tandemly coupled parts: one is a peptide synthesized by mimicking the linear epitope sequence for MAb 6-15C4 and the other is an N proteinmimicked peptide which has been shown to work as a helper T cell-stimulating antigen. Wethank Dr. R.C. Mulliganfor his kind permissionto use a retroviral expressionvector pZIP-NeoSV(X)l;Dr. Nobutaka Fujii for his help in the oligopeptidesynthesis. This work was supportedin part by a grant from the Chemo-Sero-Therapeutic Research InstituteKikuchiLaboratories(J. Nonaka, director), Kyokushi,Kikuchi-gun,Kumamoto,Japan. References 1) Bunschoten,H., Gore, M., Claassen,I.J.T.M.,Uytdehaag, F.G.C.M.,Dietzschold,B., Wunner,W.H.,and Osterhaus, A.D.M.E.1989.Characterization of a new virusneutralizing epitopethat denotesa sequentialdeterminanton the rabies virusglycoprotein.J. Gen. Virol.70: 291-298. 2) Cepko,C.L., Roberts,B.E., and Mulligan,R.C. 1984.Constructionand applicationof a highly transmissiblemurine retrovirusshuttlevector.Cell 37: 1053-1062. 3) Cox,J.H., Dietzschold,B., and Schneider,L.G. 1977.Rabies virusglycoprotein.11.Biologicaland serologicalcharacterization.Infect.Immun.16: 754-759. 4) Dietzschold,B., Gore,M., Marchadier,D., Niu, H.-S., Bunschoten,H.M., Otvos, L., Jr., Wunner,W.H.,Ertl, H.C.J., Osterhaus,A.D.M.E.,and Koprowski,H. 1990. Structural and immunologicalcharacterizationof a linearvirus-neutralizing epitope of the rabies virus glycoproteinand its possibleuse in a syntheticvaccine.J. Virol.64: 3804-3809. 5) Dietzschold,B., Wunner,W.H.,Wiktor,T.J., Lopes,A.D., Lafon, M., Smith,C., and Koprowski,H. 1983. Characterizationof an antigenicdeterminantof the glycoproteinthat correlates with pathogenicityof rabies virus. Proc. Natl. Acad. Sci. U.S.A. 80: 70-74. 6) Galfre, G., Milstein, C., and Wright, B. 1979. Rat X rat hybridmyelomasand a monoclonalanti-Fdportionof mouse IgG. Nature277-.131-133.
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M.,
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