Download Characterization of Major Structural Proteins of Measles

1397
J. gen. Virol. (1985), 66, 1397 1409. Printed in Great Britain
Key words: measles rirus/structural proteins/monoclonal antibodies
Characterization of Major Structural Proteins of Measles Virus with
Monocional Antibodies
By T. A . S A T O , *
A. FUKUDA
AND A. S U G I U R A
Department of" Measles Virus, National Institute o f Health, Gakuen 4-7-1, Musashimurayama,
Tokyo 190-12, Japan
(Accepted 28 February 1985)
SUMMARY
We have prepared and characterized monoclonal antibodies against five major
structural proteins, i.e. the HA, P, NP, F and M proteins, of measles virus. At least
three non-overlapping antigenic sites were delineated on the H A protein, three on the
P, four on the NP, four on the F and five on the M proteins by competitive binding
assays. Antigenic sites on the H A and F proteins roughly represented functional
domains defined by serological tests. The reactivity of monoclonal antibodies with
various measles virus strains including those from subacute sclerosing panencephalitis
(SSPE) and other members of the morbillivirus family was studied by immunofluorescence. A monoclonal antibody or set of monoclonal antibodies to each of the
antigenic sites showed a characteristic pattern of cross-reactivity with heterologous
strains. The H A and N P proteins were antigenically the most variable, followed by the
F and M proteins, while the P protein was relatively stable. None of the 14 anti-M
monoclonal antibodies reacted with non-virus-producing SSPE cells, strongly
suggesting the absence of M protein in these cells.
INTRODUCTION
Monoclonal antibodies have proven to be powerful tools in the study of measles virus, having
revealed subtle antigenic difference and variability of measles virus proteins previously
considered to be invariable (Birrer et al., 1981a, b; G i r a u d o n & Wild, 1981; Trudgett et al.,
1981; ter Meulen et al., 1981 ; Sheshberadaran et al., 1983), the presence of multiple antigenic
sites within some structural proteins (Carter et al., 1982, 1983a) and localization o f various viral
antigens in productive and non-productive infections (Norrby et al., t982; Johnson et aL, 1982;
Giraudon et al., 1984). We have also attempted to prepare monoclonal antibodies against
structural proteins of measles virus. Thus far, we have obtained and characterized 49
monoclonal antibodies reacting with the five major structural proteins, H A , P, NP, F and M.
The present study was concerned mainly with (i) delineation of non-overlapping sites on each
protein with monoclonal antibodies, (ii) biological functions of a n t i - H A and anti-F antibodies,
and (iii) reactivity of antibodies in immunofluorescence with heterologous virus strains
including those derived from subacute sclerosing panencephalitis (SSPE) and other members of
the morbillivirus family.
METHODS
Virus strains. Toyoshima (TY, Toyoshima et al., 1959) and Edmonston strains were used as conventional
measles virus, Hall~ and Mantooth strains as productive SSPE virus, and Onderstepoorte strain of canine
distemper virus (CDV) and LA strain of rinderpest virus (RPV) as other morbilliviruses. All these strains were
propagated in Vero cells except TY strain which was grown in KB cells. Cultures of both cell lines were
maintained in Eagle's MEM supplemented with 5~ calf serum after virus infection. The non-productive SSPE
strains were Niigata-l (N-l, Doi et al., 1972), Biken (Bik, Ueda et al., 1975), ZH and SI (Mirchamsy et al., 1978).
These strains were propagated by subculturing infected cells with flesh Vero cells.
Immunizing materials. Virions of TY strain released into culture fluid and those liberated from infected cells by
freezing and thawing were combined and purified by banding twice by centrifugation through sucrose gradients.
0000-6499 © 1985 SGM
1398
T . A . SATO, A. FUKUDA AND A. SUGIURA
Purified virions were disrupted by treatment with 2 ~ octyl-D-glucoside (OG) and 1.5 M-KCI in 0.1 i-Tris-HC1
buffer (pH 7.4) at room temperature for 1 h followed by dialysis at 4 °C for 48 h to remove OG. Viral cores were
pelleted from virions disrupted as described above, but without KCI, by centrifugation at 100000 g for 60 min. F
protein was enriched from the resultant supernatant by an immunoadsorbent column prepared as follows.
Monoclonal antibodies reacting with the HA, P, NP and M proteins (A26, A144, B11 and A27, respectively,
which are described below) were either coupled to Protein A-Sepharose CL-4B or covalently attached to cyanogen
bromide-activated Sepharose 4B. The supernatant of disrupted virus was passed through the immunoadsorbent
column and the fraction not bound to the column was collected and concentrated by lyophilization as the F
protein-enriched material. Enrichment was more efficient with cyanogen bromide-activated Sepharose than with
Protein A-Sepharose as immunoadsorbent column (data not shown).
Production ofhybridoma cell lines. Each of the four BALB/c mice was immunized by a different protocol (Table
1). Mouse A received intraperitoneal injection of OG-disrupted whole virus, whereas mice C and D were given the
F protein-enriched fraction, both emulsified in Freund's complete adjuvant. Mouse B was given native virions
without adjuvant by the same route. The animals were boosted intraperitoneally 3 to 4 weeks later with the same or
similar materials as used for priming but without adjuvant, and were sacrificed 3 days after the second injection.
Spleen cells were fused with SP2/OAgl4 myeloma cells by the method described by Kennett (1980). After
cultivation for 14 days, culture fluids were screened for antibody by ELISA with OG-disrupted virions attached to
the solid phase. Antibody-positive hybridoma cell lines were subjected to cloning by colony formation in semisolid agar and finally inoculated intraperitoneally into pristane-primed BALB/c mice. Ascitic fluid was collected 2
to 4 weeks later. The isotype of an antibody was determined by testing either ascitic fluid diluted 1 : 10 or a 10-fold
concentrate of hybridoma culture fluid by immunodiffusion against a set of rabbit antisera directed against mouse
immunoglobulins (Nordic Immunological Laboratories, Tilburg, The Netherlands).
Radioimmunoprecipitation and polyacrylamide gel electrophoresis. HeLa cells infected with TY strain at a
multiplicity of l p.f.u./cell served as the source of antigen. At 40 h, the culture fluid was replaced with leucine-,
valine- and tyrosine-free Eagle's MEM containing 50 ~tCi/ml each of [3H]leucine, [3H]valine and [3H]tyrosine
(Amersham Japan). After a labelling period of 10 h, a cell lysate was prepared as described by Sato et al. (1981 a),
except for the inclusion of 100 Kallikrein units/ml aprotinin in the lysis buffer. The cell lysate containing 1 x 106
to 5 x 106 c.p.m, was mixed with 10 ixl of ascitic fluid diluted 1 "10. Immunoprecipitation, gel electrophoresis in
10~o polyacrylamide slab gel and fluorography were carried out as described by Sato et al. (1981a).
ELISA. The procedures were similar to those described previously (Sakata et al., 1984). Purified and OGdisrupted measles virus was attached to the solid phase at a concentration of 5 ~tg/ml. The peroxidase-conjugated
IgG fraction of rabbit anti-mouse IgG serum (Cappel Laboratories, Cochranville, Pa., U.S.A.) was used for
detection of anti-measles virus antibody in hybridoma culture fluids.
Competitive binding ELISA. Peroxidase was conjugated to IgG precipitated by ammonium sulphate from ascitic
fluid by the method of Wilson & Nakane (1978). The dilution of conjugate to give an absorbance value of
approximately 1.0 in direct ELISA was determined. Procedures for ELISA were similar to those described above.
A conjugate at predetermined concentration was mixed with various dilutions of an unlabelled antibody. The
mixtures were allowed to react with the antigen-coated wells for 2 h at room temperature. The subsequent steps
were carried out as described previously (Sakata etal., 1984).
Serological tests. Haemagglutination inhibition (HI) and haemolysis inhibition (HL[) tests were carried out by
the methods described by Norrby & Gollmar (1972). Neutralization (NT) tests were by plaque assay on Vero cells
grown in Costar 12-well cluster plates (Fukuda & Sugiura, 1983). The highest dilution that caused 50% plaque
reduction was taken as the NT titre.
Immunofluorescence. Either Vero or HeLa cells grown on coverslips were infected with various virus strains.
When cytopathic effect developed to a moderate degree, cells were subjected to immunofluorescent staining either
without fixation or after fixation with acetone for 20 min at - 20 °C. Cells infected with non-productive SSPE
strains were used 2 to 3 days after seeding. Monoclonal antibodies were tested at serial dilutions, usually at 1 : 30,
1 : 100, 1 : 300, 1 : 1000 and 1 : 3000. Fluorescein conjugate of rabbit anti-mouse IgG (Cappel Laboratories) was used
at a dilution of 1:50. Coverslips were mounted in 50% glycerol in phosphate-buffered saline and examined by
epifluorescence with a Nikon microscope.
RESULTS
Preparation and specificity o f monoclonal antibodies
F o r t y - n i n e c l o n e s o f h y b r i d o m a s s e c r e t i n g m o n o c l o n a l a n t i b o d i e s r e a c t i n g w i t h m e a s l e s virus
p r o t e i n s w e r e d e r i v e d f r o m four mice. H y b r i d o m a s w e r e i n o c u l a t e d i n t o t h e p e r i t o n e a l c a v i t y o f
s y n g e n e i c m i c e a n d the ascitic fluids collected w e r e used as m o n o c l o n a l a n t i b o d i e s . T h e
specificity o f t h e s e a n t i b o d i e s w a s d e t e r m i n e d by r a d i o i m m u n o p r e c i p i t a t i o n a n d S D S - P A G E
(Fig. 1). A n t i b o d i e s w e r e d e s i g n a t e d by t h e m o u s e f r o m w h i c h h y b r i d o m a s w e r e d e r i v e d (A, B,
1399
Monoclonal antibodies to measles virus proteins
(0
U)
(k)
(l)
(m)
(
(a)
(b)
(c)
(d)
(e) (f)
(g)
(h)
200K ~92.5K•
HA
P
69K •
NP
46K •
II
F1
M
30K•
Fig. 1. SDS-PAGE fluorogram of measles virus proteins precipitated by monoclonal antibodies from
measles virus-infected Vero cell lysates. (a) A2(HA); (b) B5(HA); (c) A144(P); (d) A56(NP); (e)
B1 I(NP); 0'; i) serum from atypical measles case; (g) C527(F); (h) D84(F); (j) A23(M); (k) A27(M); (/)
AI54(M); (m) AI77(M).
Table 1. Hybridoma clones obtained from mice immunized with Toyoshima ( TY) strain of measles
virus
No. of hybridoma clones producing
antibody against
A
(
Mouse
A
B
C
D
Primed with
OG-disrupted
virus with
adjuvant
Native virions
F-enriched
material*
with adjuvant
F-enriched
material*
with adjuvant
Boosted with
OG-disrupted
virus
HA
2
P
1
NP
3
F
0
M
13
Total
19
Native virions
F-enriched
material*
7
2
0
2
3
0
1
5
1
0
12
9
F-enriched
materiaH"
0
0
0
9
0
9
14
49
Total
11
3
6
15
* Enriched with Protein A-Sepharose immunoadsorbent column.
t Enriched with cyanogen bromide-activated immunoadsorbent column.
C and D) followed by a n u m b e r and, when necessary, the specificity in parentheses. Table 1
summarizes the n u m b e r of hybridomas of different specificity derived from individual mice.
Different immunization protocols resulted in different spectra of hybridomas in terms of
specificity as reported previously (Bohn et al., 1982). The mouse immunized with native virions
(mouse B) yielded mainly hybridomas specific to H A and NP, while those specific to internal
proteins predominated when the animal was given the detergent-disrupted material. Most antiF hybridomas were derived from the mice given materials enriched for F protein.
1400
T. A . S A T O ,
I
A. FUKUDA
I
I
[
I
(a)
AND
b~
A. S U G I U R A
I
I
I
I
)?
@
"~ 100
50
I
-1
I
I
i
I
J
- 1 --2 - 3 - 4 --5
2 -3 -4 -5
log,0 Dilution of unlabelled antibody
Fig. 2. Competitive IELISAof monoclonal antibodies directed against the P protein. Absorbance
values developed by a peroxidase-conjugatedanti-P antibody when mixed with unlabelled antibodies at
the indicated dilutions and applied to the antigen on solid phase were expressed as a percentage of the
absorbance value in the absence of unlabelled antibody. Peroxidase-conjugatedantibody: (a) A 144(P);
(b) C6(P). Unlabelled antibody: O, AI44(P): A, C6(P); I , CII0(P); C), A56(NP).
Delineation of non-overlapping antigenic sites by competitive antibody binding
Antigenic sites to which the monoclonal antibodies bound were analysed by competitive
binding assays. Binding of peroxidase-conjugated antibodies to the solid phase antigen was
determined in the absence and presence of various concentrations of unlabelled antibodies.
Delineation of non-overlapping antigenic sites by this method was, in general, unambiguous.
Only homologous antibodies effectively competed with binding of A 144(P) and C6(P). Other
antibodies, including A56(NP), had little effect upon the binding (Fig. 2). The epitopes
recognized by A144 and C6 were therefore considered to represent two different antigenic sites
of P protein. Another antibody, C110(P), competing with neither, was thought to react with a
third antigenic site. Thus, at least three non-overlapping antigenic sites (I, II and III) are present
on the P protein (Table 5). Four non-overlapping antigenic sites were differentiated on the NP
protein (Fig. 3, Table 5). B4 and BI 1 behaved like BI in a competition test (data not shown). Site
II recognized by A67 was blocked by antibodies reacting with site I (A49) or site III (B 1, B4 and
Bll) but A67 did not at all inhibit the binding of the latter antibodies (Fig. 3). Such
unidirectional interaction may result from the conformational change of site II induced by the
attachment of antibody at a distant site. Results of competitive binding assay with
representative anti-M monoclonal antibodies are shown in Fig. 4 and are summarized in
Table 5. Five non-overlapping antigenic sites of M protein have been delineated. The
competition between representative anti-HA monoclonal antibodies is depicted in Fig. 5 and
the overall results are summarized in Table 2. Each of the labelled antibodies was competed by
heterologous antibodies to various degrees and, although competition was not always reciprocal,
three non-overlapping or partially overlapping antigenic sites (I, II and III) could be roughly
defined. One-way competition was more frequent between antibodies against the HA protein
than between those against other proteins. It may reflect the ease with which the HA protein is
1401
Monoclonal antibodies to measles virus proteins
]
I
I
I
I
]
I
I(a)
t
I
I
I
I
I
I
I
I
I
I
. (c)
](b)
I
I
I
I
(d)
"9 100
i
50[
-12-3-4-5
-1 2 - 3 - 4 - 5
-1 - 2 -3 - 4 5
lOgl0Dilution of unlabelled antibody
Fig. 3. Competitive EEISA of monoclonal antibodies directed against the NP protein (see Fig. 2).
Peroxidase-conjugated antibody: (a) A49(NP); (b) A67(NP); (c) BI(NP); (d) A56(NP). Unlabelled
antibody: O, A49(NP); A, A67(NP);., BI(NP); O, A56(NP); A, A144(P).
I
i
I
I
1
1
-1-2-3-4-
1
1
I
~
I
l
I
i
I
1
1
1
1
~
1
1
1
~
]
1
1
1
1
1
I:',
II
I
-1-2
I
I
I
t
I
I
I
3--4 5
1 2 - 3 4--5 - 1 - 2 - 3 - 4 - 5
1-2-3-4-5
1 2 - 3 - 4 -5
log ~0Dilution of unlabetled antibody
Fig. 4. Competitive ELISA of monoclonal antibodies directed against the M protein (see Fig. 2).
Peroxidase-conjugated antibody : (a) A 184(M); (b) A27(M) ; (c) A51(M); (d) A 133(M); (e) A 137(M).
Unlabelled antibody: O, AI84(M); A, A27(M); II, A5I(M); O, AI33(M);/h, AI37(M); [], A144(P).
subject to conformational change. Competition of three anti-F monoclonal antibodies by
homologous and representative heterologous antibodies is shown in Fig. 6. The results of
competitive binding assay by all anti-F monoclonal antibodies are summarized in Table 3. Each
of the three labelled antibodies was shown to represent a non-overlapping antigenic site.
Antibody D122 did not compete with any of the three labelled antibodies and was therefore
thought to be directed against a putative fourth antigenic site.
1402
r.A.
SATO, A. FUKUDA AND A. SUGIURA
150
O
e~
r"
100
.~_
,_
~
50
- 1 --2 --3 -4
-5
-- I--2 -3
--4 -5
- 1 --2 --3 -4
--5
- 1-2
-3
-4
-5
log ~0Dilution of unlabelled antibody
Fig. 5. Competitive ELISA of monoclonal antibodies directed against the HA protein (see Fig. 2).
Peroxidase-conjugated antibody: (a) B5(HA); (b) B12(HA); (c) A2(HA); (d) C146(HA). Unlabelled
antibody: O, B5(HA); A, B12(HA); II, A2(HA); O, C146(HA); A, A56(NP).
T a b l e 2.
Summary of competitive binding ELISA of anti-HA monoclonal antibodies*
l-
Peroxidase-labelled antibodies
Unlabelled
antibody
B5
B67
B69
B71
B7
BI2
B2
A26
A2
C46
CI46
Antigenic
site
I
1
I
I
1
I
I&ll
ll
II
III
llI
•
A
B5
+ +
+ +
+ +
+ +
+
+ +
+ +
B69
++
++
++
++
+
++
++
B12
++
++
++
++
++
++
++
_
_
+
-
_
+
-
B2
++
++
++
++
-++
++
++
A26
i
A2
+
+
+
C146
+
++
++
+
-
++
-
++
* + +, Complete inhibition; +, partial inhibition; - , no inhibition.
Biological activities of monoclonal antibodies
N o n e o f the anti-P, a n t i - N P and a n t i - M m o n o c l o n a l a n t i b o d i e s had H I , H L I or N T activity.
O n the o t h e r hand, m o s t a n t i b o d i e s r e a c t i n g with a n t i g e n i c sites I and II o f H A possessed these
activities (Table 4). N T activity relative to H I a c t i v i t y was g r e a t e r for a n t i b o d i e s d i r e c t e d
against site I t h a n for those directed against site II. T h i s finding suggests that w h e r e a s the two
antigenic sites are equally i n v o l v e d in h a e m a g g l u t i n a t i o n , site I plays a m o r e i m p o r t a n t role in
the initiation of infection t h a n site II. A n t i b o d i e s d i r e c t e d against site I I I had no H I , H L I or N T
activity. A n t i g e n i c sites defined by c o m p e t i t i v e b i n d i n g assay thus a p p e a r e d to r e p r e s e n t
functional d o m a i n s o f the H A molecule.
Monoclonal antibodies to measles virus proteins
1403
O
•~
100
..Q
.=_.
~
50
--1-2-3--4-5
-1-2--3--4--5
--1--2-3--4-5
log~0 Dilution of unlabelled antibody
Fig. 6. Competitive ELISA of monoclonal antibodies directed against the F protein (see Fig. 2).
Peroxidase-conjugated antibody: (a) D43(F); (b) C527(F); (c) Dll4(F). Unlabelled antibody: O,
D43(F); A, C527(F); I I , D I I 4 ( F ) ; O, A56(NP).
T a b l e 3.
Summary of competitive binding ELISA of anti-F monoclonal antibodies*
Unlabelled
antibody
Antigenic
site
B48
C55
CII1
Cl13
C177
C183
D43
D240
C527
D84
D114
D134
D135
D148
D122
I
I
I
I
I
I
I
I
II
III
III
III
III
III
IV
Peroxidase-labelled antibodies
~
~
D43
C527
D 114
+ +
+ +
++
++
+ +
+ +
+ +
+ +
+
-
+ +
-
+
+
+
+
+
+
+
+
+
+
-
* + + , Complete inhibition; + , partial inhibition; - , no inhibition.
Anti-F monoclonal antibodies had no HI, HLI or NT activity except antibody C527 directed
a g a i n s t a n t i g e n i c site I I o f t h e F p r o t e i n w h i c h i n h i b i t e d v i r u s - i n d u c e d h a e m o l y s i s a n d
n e u t r a l i z e d i n f e c t i v i t y a t l o w d i l u t i o n s ( T a b l e 4). S i n c e C 5 2 7 w a s t h e o n l y a n t i b o d y t h a t
r e c o g n i z e d site I I , it is n o t k n o w n w h e t h e r t h i s site is t h e d o m a i n o f t h e F p r o t e i n p a r t i c u l a r l y
important for the fusion activity.
1404
T. A. SATO, A. FUKUDA AND A. SUGIURA
T a b l e 4. Serological tests with anti-HA and anti-F monoclonal antibodies
Antibody
B5(HA)
B67(HA)
B69(HA)
B71(HA)
B7(HA)
B12(HA)
B2(HA)
A26(HA)
A2(HA)
C46(HA)
C146(HA)
B48(F)
C55(F)
Clll(F)
CI13(F)
C177(F)
C183(F)
D43(F)
D240(F)
C527(F)
D84(F)
D114(F)
D134(F)
D135(F)
D148(F)
DI22(F)
Isotype
G2a
G2a
G2b
G2b
G2a
G1
G2a
G1
G1
G2a
G2b
G2a
GI
GI
GI
G1
GI
G2a
G2b
G2b
G2a
G2a
G2a
G2a
G2a
G2a
Antigenic
site
I
I
I
I
I
I
I & I!
II
II
Ill
III
I
I
I
I
I
I
I
I
II
111
III
III
III
III
IV
•
HI*
20480
20 480
640
1280
25 600
320
12800
25 600
2560
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
Antibody titre
x
HLIt
640
320
< 10
< 10
160
160
160
80
80
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
40
< 10
< 10
< 10
< 10
< 10
< 10
..
NT~
640000
128 000
32 000
128000
4000
160
16000
640
40
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
< 10
40
< 10
< 10
< 10
< 10
< 10
< 10
* HI, Haemagglutination inhibition test.
t HLI, Haemolysis inhibition test.
~:NT, Virus neutralization test.
Immunoreactivity of monoclonal antibodies with various strains of morbillivirus
All m o n o c l o n a l a n t i b o d i e s used in this study reacted with acetone-fixed V e t o cells infected
with T Y strain in indirect i m m u n o f l u o r e s c e n c e . A n t i b o d i e s against the H A and F proteins also
stained the surface of unfixed cells, while a n t i b o d i e s directed against the P, N P and M proteins
failed to react with unfixed cells, i n d i c a t i n g the absence of these internal proteins at the cell
surface. T h e reactivity o f m o n o c l o n a l a n t i b o d i e s w i t h heterologous strains was studied by
i m m u n o f l u o r e s c e n t staining of acetone-fixed V e r o cells infected with various strains o f measles
virus including S S P E strains and o t h e r m e m b e r s o f the m o r b i l l i v i r u s family. T h e a n t i b o d i e s
were applied at five dilution steps r a n g i n g f r o m 1:30 to 1:3000. T h e dilution e n d p o i n t of a
particular a n t i b o d y is g i v e n by the score - , + and + + in T a b l e s 5, 6 and 7. T h e score
represents, therefore, the rough q u a n t i f i c a t i o n o f the reactivity o f a p a r t i c u l a r a n t i b o d y w i t h the
c o r r e s p o n d i n g antigen of a g i v e n virus strain. T h e intensity of fluorescence, n a m e l y the a m o u n t
of the antigen present, was not t a k e n into account. T h e only e x c e p t i o n was the M protein of nonproducing S S P E ceils where M protein was m o r e likely to be absent t h a n to be antigenically
altered as discussed below. In general, a set of m o n o c l o n a l a n t i b o d i e s reacting w i t h one
antigenic site had a very similar, if not identical, r e a c t i v i t y pattern w i t h heterologous strains.
T h r e e a n t i @ a n t i b o d i e s reacted w i t h all measles virus strains. T w o of t h e m r e a c t e d also w i t h
both C D V and R P V , but one (A144) was specific to measles virus, cross-reacting w i t h n e i t h e r
C D V nor R P V . M o r e c o m p l e x p a t t e r n s were o b s e r v e d for a n t i - N P antibodies. T h e e p i t o p e
recognized by A56 was c o m m o n to all strains e x a m i n e d . A n t i b o d i e s reacting w i t h the three
other antigenic sites r e v e a l e d antigenic differences b e t w e e n p r o d u c t i v e measles virus, nonp r o d u c t i v e S S P E virus strains, C D V and R P V and b e t w e e n n o n - p r o d u c t i v e S S P E strains
themselves. T h r e e reactivity p a t t e r n s were found w i t h a n t i - M antibodies. A n t i b o d y directed
Monoclonal antibodies to measles virus proteins
T a b l e 5.
1405
Immunoreactivity* of monoclonal antibodies against three internal proteins of measles virus
in indirect immunofluorescence with different strains of morbillivirust
SSPE
Measles
~
~
TY
ED
,
Productive
~
~
Ha
Man
'ZH
++
+ +
+ +
+ +
+ +
+ +
++
+ +
+ +
+ +
+ +
+ +
++
+ +
+ +
+ +
+ +
+ +
++
+ +
+ +
+
+ +
+ +
++
+ +
+ +
.
-
++
+ +
+ +
+ +
.
-
+ +
NT~
+ +
NT
--
Antibody
Isotype
Antigenic
site
AI44(P)
C6(P)
Cll0(P)
A49(NP)
A67(NP)
BI(NP)
B4(NP)
Bl I(NP)
A56(NP)
A184(M)
A23(M)
A24(M)
A27(M)
A154(M)
A157(M)
A177(M)
B46(M)
A39(M)
A41(M)
A42(M)
A51(M)
A133(M)
A 137(M)
GI
G2a
G2a
G2a
G1
G2a
G2a
I
II
III
I
II
III
III
G2b
III
+ +
NT
G1
GI
G2a
G2a
G1
Gl
GI
G1
G2a
G1
G1
G2a
GI
GI
G1
IV
I
II
II
II
II
II
II
II
III
III
III
III
IV
V
+ +
+ +
+ +
+ +
+ +
+ +
+ +
++
+ +
+ +
+ +
+ +
+ +
++
+ +
+ +
+ +
+ +
+
+ +
+ +
+ +
++
NT
+ +
+
NT
+
++
+ +
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+ +
+
+
+ +
+ +
++
+ +
Non-productive
x
SI
N-l
++
+ +
+ +
+
+ +
++
+ +
+ +
+
.
+ +
--
+ +
+ +
.
.
Others
r~'
CDV
RPV
~
Bik
+ +
+ +
+
+ +
+ +
+ +
.
-
-
--
--
NT
--
--
+ +
+ +
--
--
+ +
+ +
+ +
+
+ +
+ +
+
++
+ +
+ +
+ +
+
+
++
+
+ +
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+ +
+ +
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+ +
+ +
+
+ +
+ +
+ +
+ +
+ +
++
+
+ +
+ +
-+
+
+ +
+
+
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
++
.
* Dilution endpoint: + +, >/1:1000; +, 1:30to1:300;
-, <1:30.
t TY, T o y o s h i m a ; ED, E d m o n s t o n ; Ha, Hall6; M a n , M a n t o o t h ; N - I , N i i g a t a - 1 ; Bik, B i k e n ; C D V , c a n i n e
d i s t e m p e r virus; R P V , r i n d e r p e s t virus.
:~ NT, Not tested.
T a b l e 6.
Immunoreactivity* ojanti-HA monoclonal antibodies in indirect immunofluorescence with
different strains of morbillivirus~f
SSPE
A
Measles
Antibody
Antigenic
site
rT Y
B5
B71
B7
BI2
B2
A26
A2
C46
I
I
I
I
I & II
II
II
11I
++
++
++
++
+ +
+ +
++
++
h ED ~
++
++
++
++
.
+ +
++
++
Productive
cHa
J'
++
++
++
++
.
+
++
++
Non-productive
~
Man
~
Z
H
SI
++
++
++
++
++
++
++
++
.
+
++
++
++
++
++
++
.
.
.
++
.
.
+
++
++
--~
N-I
Bik
++
++
++
++
++
++
++
++
.
.
.
.
.
.
++
* D i l u t i o n e n d p o i n t : + + , ~>1:1000; + , 1:30 to 1:300; - ,
t For m o r b i l l i v i r u s strains, see T a b l e 5.
against
antigenic
antigenic
antigenic
site I cross-reacted
sites II and
with both
IV cross-reacted
sites III and V cross-reacted
with
CDV
CDV
with RPV
Others
~
c-'-~
CDV
RPV
-
-
-
-
.
.
.
++
.
.
<1:30.
and
RPV.
Antibodies
directed
against
only,
while
antibodies
directed
against
only. No antigenic
difference of P, NP and M
proteins was found among conventional measles virus strains and virus-producing SSPE strains.
Cross-reactivity
of antibodies against the HA and F proteins with heterologous strains is
summarized
in Tables
6 a n d 7, r e s p e c t i v e l y .
Epitopes
on antigenic
sites I and III of the HA
1406
T.A.
SATO,
A. F U K U D A
AND
A. SUGIURA
Table 7. Immunoreactivity* of anti-F monoclonal antibodies in indirect immunofluorescence with
different strains o f rnorbillivirus~
SSPE
A
r
Antibody
Clll
D43
C527
D135
D148
D122
Antigenic
site
I
I
II
III
III
IV
Measles
~"
cTy
ED ~
Productive
r
"~
,
Ha
Man
c
ZH
++
++
++
++
+ +
++
++
++
++
++
+ +
++
++
++
++
++
+ +
+
++
++
++
++
+ +
++
++
++
++
++
+ +
++
Non-productive
~
SI
N-1
++
++
++
+
++
++
++
++
+
+
* Dilution endpoint: ++,
~>1:1000; + , 1 : 3 0 to 1 : 3 0 0 ; - ,
t F o r m o r b i l l i v i r u s s t r a i n s , see T a b l e 5.
~
Bik
++
++
++
-
Others
' ~"
CDV
RPV
++
++
++
+ +
++
++
++
++
++
+ +
++
<1:30.
protein appeared to be well conserved among all measles virus strains including the SSPE virus
strains, with the exception of the epitope recognized by antibody B2 which was located where
sites I and II overlap and was missing in all strains other than the TY strain. Epitopes on site II
were more variable, being lost by some non-productive SSPE virus strains. None of the HA
antibodies so far tested cross-reacted with either CDV or RPV. Antibodies against sites I and II
of the F protein reacted with all measles virus strains while those against site III and IV failed to
react with some of the non-productive SSPE virus strains. Most epitopes of the F protein, in
contrast to those of the HA protein, were shared by other morbilliviruses except the one
recognized by antibody C527 which was missing in the F protein of CDV. All monoclonal
antibodies to the HA or F proteins so far obtained by us stained acetone-fixed as well as unfixed
infected cells. The earlier studies had shown that acetone destroys the antibody-binding activity
of the haemolysin (Fraser et al., 1978; Armstrong et al., 1979). The exact nature of the
haemolysin has yet to be determined. Should it be identical with either the HA or F proteins, the
above reports would indicate the existence of a still unidentified acetone-labile epitope(s) in
either of these proteins.
DISCUSSION
We have defined non-overlapping antigcnic sites by competitive binding assay for each of the
five measles virus structural protcins. Similar attempts had been made by previous investigators
with rcspect to the HA and M proteins (tcr Meulcn et al., 1981; Carter et al., 1982, 1983a;
Sheshbcradaran et al., 1983), but not for other proteins. A significant feature was thc rclcvance
of antigenic sites thus defined to the antigenic and biological properties of a protein. First,
antigenic sites on the HA and F proteins roughly represented functional domains of cach
protein. The results obtained for the HA protein were, in general, comparable to those in carlier
rcports (ter Meulen eta/., 1981; Carter et al., 1982). In view of the similarity of biological
properties, antigenic sites I, II and III of TY strain appeared to be analogous to binding groups
3, 2 and 1, respectively, of Edmonston strain (Carter et al., 1982). Secondly, a monoclonal
antibody or a set of monoclonal antibodies assigned to each of the antigenic sitcs showed a
characteristic reactivity pattern with heterologous virus strains and the pattern was very similar,
if not identical, within the set. Two interpretations are possible for the latter finding. First, all
antibodies within a set might have been the products of hybridomas derived from antibodyproducing spleen cells of a single lineage and, thus, directed against an identical antigenic
determinant. The possibility does not seem plausible, however, in view of the diverse subclasses
of these antibodies. Alternatively, and more likely, most antigenic differences between strains
may have resulted from multiple changes within an antigenic site. This interpretation, in turn,
leads to the speculation that the antigenic diversification of morbilliviruses occurs through
accumulation of antigenic changes clustered in certain limited regions rather than scattered
randomly throughout the polypeptides.
Monoclonal antibodies to measles virus proteins
1407
The present study showed different antigenic stability for each of the five structural proteins.
The P protein appeared to be antigenically the least variable, two out of three antigenic sites
being essentially conserved among all morbillivirus strains. This could be due to the
conformational constraints imposed on the P protein which is likely to be an essential
constituent of the transcription and/or replication machinery. By contrast, the NP and HA
proteins were more variable. Only one out of four antigenic sites on the NP and none of three
sites on the HA proteins are shared by CDV and RPV. Worthy of note is the antigenic difference
of the NP, HA and, to a lesser extent, F proteins of non-productive SSPE virus strains from
those of measles virus and productive SSPE virus strains. It has been shown that a viral genome
may accumulate more mutations during persistence than during productive infection (Holland
et al., 1979). It is intriguing to speculate that the extensive antigenic variation resulted from the
prolonged persistence of these SSPE virus strains. It may be significant, in this context, that
productive SSPE virus strains were closer in antigenicity as well as in replication pattern to
conventional measles virus than non-productive SSPE virus strains. The possibility cannot be
ruled out, of course, that these non-productive SSPE virus strains originated in progenitors
appreciably different antigenically from the conventional measles virus strains employed here.
It should also be noted that the antigenic site of the NP protein conserved in CDV and RPV (site
IV) was also conserved in non-productive SSPE virus strains. This particular site of the NP
protein might be functionally more important than others. Out of four antigenic sites of the F
protein, three sites were retained unchanged by CDV and RPV and the remaining one by RPV
only. Antigenic stability of this protein has been reported previously (Sheshberadaran et al.,
1983).
All anti-M monoclonal antibodies failed to react with non-producing SSPE cells. This is
consistent with the earlier observation by one of us that the serum from an atypical measles case
did not precipitate M protein from the cells harbouring these SSPE virus strains (Sato et al.,
1981 b), and also with the results obtained from cells infected with D R strain of non-productive
SSPE virus by immunofluorescence with polyclonal antiserum as well as monoclonal antibodies
(Johnson et al., 1982). Although the absence of an immunologically identifiable protein may
mean either the absence of the protein per se or the extensive antigenic alteration of the protein
in question, the former possibility is more compatible with a number of previous studies (Hall &
Choppin, 1979; Lin & Thormar, 1980; Machamer et al., 1981 ; Johnson et al., 1981, 1982; Carter
et al., 1983b). Although it had been reported that there was extensive variation in the M
proteins of nine measles virus strains in contrast to the relative stability of the NP and P proteins
(Sheshberadaran et al., 1983), the M proteins of both the conventional measles virus and the
productive SSPE virus strains examined in this study were antigenically uniform. Out of five
antigenic sites only one site was shared by both CDV and RPV. Two sites were retained by CDV
only and the other two by RPV only. This finding, although incomplete in the absence of
reciprocal data, is suggestive of the equidistance of measles virus from both CDV and RPV,
which may be of possible significance in the evolutionary relationship of morbilliviruses. Taken
together, among five structural proteins of measles virus, the HA and NP proteins appeared to
be most variable, while the P protein was the least variable. The stability of the M and F proteins
was intermediate.
T h e authors wish to thank Dr M. Arita for his advice in hybridoma technology, Ms H. Sakata for her help in
developing ELISA and Dr T. K o h a m a for his helpful discussion. This work was supported by Grant-in-Aid from
the Ministry of Education, Japan.
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