Journal of General Virology (1992), 73, 2211-2216. Printed in Great Britain
2211
Heterogeneity of linear B cell epitopes of the measles virus fusion protein
reacting with late convalescent sera
K.-H. Wiesmiiller, 1 G. Spahn, 2,3 D. Handtmann, 2,3 F. Schneider, 3 G. Jung 1 and C. P. Muller2'3*t
11nstitut f f i r Organische Chemie and 2Medizinische Fakulti~t, Universiti~t Ti~bingen, Germany and
3Laboratoire National de Santk, Boite Postale 1102, L - l O l l L u x e m b o u r g
B cell epitopes of the measles virus fusion protein
were mapped, by reacting sera from late convalescent
donors with synthetic overlapping pentadecapeptides,
segments covering the whole F protein sequence.
Unselected individual sera recognized 7 to 20% of the
total sequence. Cumulation of the binding patterns of
30 sera identified eight to 10 clusters of antibodybinding peptides spread over most of the sequence. The
B cell epitopes included regions of transition between
the more hydropathic (including the N-terminal end of
the F1 and F2 protein) and hydrophilic sequences.
When the regions of antibody binding were compared
with the predicted secondary structure of the F protein,
no detectable pattern became apparent. Exposed
sequences as well as sequences hidden in the viral
membrane or in the protein core of both the F1 and F2
polypeptides were recognized by the antibodies. The
heterogeneity of the binding patterns was not merely
dependent on the anti-measles virus titre. The importance of antibodies recognizing linear epitopes of the
measles virus fusion protein for the immune protection
is presently not known.
Introduction
Peptides. One-hundred and eight overlapping pentadecamer
peptides covering the whole sequence of the F protein of the measles
virus (strains Edmonston and Hall& Richardson et al., 1986; Buckland
et aL, 1987)were synthesized on a multiple peptide synthesizer (Zinsser
SMPS 350; Zinsser Analytics). p-Benzyloxybenzyl alcohol resin
(30 mg) loaded with the first Fmoc-amino acid was filled in separate
small tubes (Eppendorf cups). Fmoc deprotection was carried out with
50~ piperidine in N,N-dimethylformamide (0.5 ml) for 5 min and
repeated for 15 min. Single couplings were performed with Fmocamino acids in 10-foldexcess and 1-hydroxybenzotriazole/diisopropylcarbodiimide activation in N,N-dimethylformamide within 1 h. After
coupling and after deprotection the tubes were washed with
N,N-dimethylformamide (0.5 ml), 10 times each. N-Fmoc- and side
chain-protected pentadecapeptide resins were washed with dichloromethane, N,N-dimethylformamide and methanol, dried in vacuo and
treated with 0.5 ml of triftuoroacetic acid:thiocresol:thioanisole
(95:2:3) for 3 h. The NoFmoc peptides were washed twice by
sonication in ether. The precipitates were dissolved in tertiary butanol
and this solution was lyophilized. Peptide samples were dissolved in
6 M-HCIand hydrolysedfor 24 h at 110 °C. Amino acids were separated
as trifluoroacetyl-aminoacid propylesters by gas chromatography on a
glass capillary coated with ChirasiI-Val. The D-amino acid content was
determined by enantiomer labelling (Frank et al., 1978). Amino acid
analysis produced results according to the expected data, and no
racemization could be detected. The peptides were designated by
runningnumbers starting with the C-terminal peptide KPDLTGTSKSYVRSL. One-hundred and four peptides overlap with 10 amino acids
with their adjacent peptides (Fig. 1). For chemical reasons, peptides 21,
41, 91 and 99 were shifted by one amino acid position towards the N
terminus and overlapped by nine amino acid residues with the previous
peptides.
A n t i b o d i e s play a n i m p o r t a n t p a r t i n i m m u n e p r o t e c t i o n
a g a i n s t measles. This is highlighted in n e w b o r n a n d
i n f a n t c h i l d r e n who are protected by t r a n s p l a c e n t a l l y
acquired m a t e r n a l a n t i b o d i e s (Albrecht et al., 1977;
Black, 1989). T h e protective role of a n t i b o d i e s is
confirmed by the successful p r e v e n t i o n of measles
o u t b r e a k s with infusions of i m m u n o g l o b u l i n s with
elevated anti-measles virus activity (Janeway, 1945).
T w o m e m b r a n e proteins of the measles virus, the
h a e m a g g l u t i n i n (HA) a n d the fusion (F) p r o t e i n are
k n o w n to m e d i a t e a protective i m m u n e response
( G i r a u d o n & Wild, 1985; M a k e l a et al., 1989). T h e r e is
e v i d e n c e that a h u m o r a l response to the F p r o t e i n is
required for complete p r o t e c t i o n ( N o r r b y et al., 1975;
Merz et al., 1980). I n the present study, the authors
m a p p e d the l i n e a r a n t i b o d y - b i n d i n g sites of the measles
virus F p r o t e i n using o v e r l a p p i n g synthetic p e n t a d e c a peptides covering the whole sequence.
Methods
Sera. Sera were collected from 30 healthy adult blood donors. The
measles virus titres were determined using a commercial complement
fixation kit (Virion, CH-6330 Cham).
tAddress correspondence to: Dr Claude P. Muller, Laboratoire
National de Sant6, Boite Postale 1102, L-1011 Luxembourg.
0001-0931 © 1992 SGM
Peptide ELISA. Flat-bottomed microtitre plates (Immunoplate
Maxisorb F96; Nunc) were coated overnight at room temperature with
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K.-H. Wiesmiiller and others
2212
I0
20
~0
40
50
60
70
80
gO
100
k~F~kVLL~L~1PTG~HW~NLSK~G~G~ASYK~TRSSH~SL~IK~PN~LLNNC~RV[~A[YRRL~RIvL~P~RD~NAH~N~RP
IOB
IO~
106
I05
104
IO]
102
}gl
tO0
99
98
97
96
95
94
9~
92
91
110
120
I]0
140
150
160
170
180
190
200
~S~AS~RRHKRF~GVV~AG~LG~AT~A~T~G~S~NS~DNLRk~L~N~A~Ek~R~GQ~kV~V~YiNN~L~N~LSCD~GQ
90
89
88
87
86
85
84
83
82
81
80
79
78
77
76
75
74
73
~%0
22D
230
240
250
260
270
280
K~GLK~RYYTE~LSLFGPS~R~PiSAE~S~LSY~LG¢D~NKV~K~GYSGG~1LG~ESR~K~R~IHV~SYF~LS~AYPTLSE~KGv~HR~
BO
69
68
67
66
65
54
63
62
61
60
59
57
58
49
48
41
46
45
44
43
42
41
40
39
38
]7
36
410
420
4bO
440
450
460
470
G~ti~P~Klt]Y~AA~H~PV~N~v~GS~RYP~VY~H~GPP~S~[R~D~G~N~GNk1~Kt~A~[L~S~S~GLS~S~Y~L~
~0
29
2B
27
26
25
24
2J
22
2~
20
19
I8
17
52
51
390
]5
34
400
33
480
16
~00
53
54
310
320
330
340
550
360
~70
]BO
GVSYNrGS~wYI~P~YVA]~GY~SNF~ES~CT~P~G~vCS~N~LYPuSPL~Q~CLRGSlKSC~RTLvSGSFGNRF~S~GN~ANCAS[~CKCY~I
50
71
290
5fi
55
72
31
32
490
15
14
50Q
15
12
II
5~0
5~
5bO
b40
55O
AVCLCCLIGIPALICCCRCRCNKKGFQVGMSRPGLKPDLTCTSKSYVRSL
}0
g
B
/
6
5
4
3
2
Fig. 1. Amino acid sequence of the measles virus F protein including the signal peptide and Fmoc-pentadecapeptides used in this
study. Numbers above and below the sequence indicate amino acid positions and designations of the overlapping peptides (last digit
underneath the C-terminal end of the peptide) respectively.
50 pl double-distilled water containing 10 pg peptide. Before use the
plates were blocked for 2 h at 37 °C with 1% BSA in Tris-buffered
saline (TBS) and extensively washed with 1 ~o Tween-20 in TBS. The
plates were then incubated for 2 h at room temperature with 50 pl
serum, diluted l: 160 in 1% BSA, 0.1% Tween in TBS, washed again
and developed with 50 pl of F(ab')2 of goat anti-human IgG conjugated
to alkaline phosphatase (7 chain-specific, 1 : 500 in the above dilution
buffer; Sigma). After washing, 0.658 mg/ml p-nitrophenylphosphate
disodium salt in substrate buffer was added and the absorbance (A) was
measured after 1 to 3 h at 405 nm in a Multiskan plus (Titertek) ELISA
reader.
Binding data for individual donors are presented as mean of
duplicate A~o ~ values (Fig. 2). Owing to differences in background
intensity a direct comparison of A4os values of different donors was not
useful. Instead the data (Fig. 3) were expressed in multiples of the
background. The background was calculated using 50% of the lowest
values, as was done by others (Trifilieff et al., 1990).
Secondary structure prediction. Estimations of hydrophilicity (Kyte &
Doolittle, 1982) and hydropathy (Hopp & Woods, 1981 ) were combined
with acrophilicity (Hopp, 1981) and conformational predictions
(Garnier et al., 1978).
(a)
1200
I
I
I
I
I
I
I
1
I
I
I
I
I
I
I
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800
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itlhlhlllll6.
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.
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B&ding of unselected sera
]
Sera from healthy donors with anti-measles virus
antibody titres ranging from 1:4 to 1 : 128 were reacted
with immobilized Fmoc-pentadecapeptide conjugates in
an ELISA. Fig. 2 shows the reaction of two individual
sera (no. 176, titre 1 : 128 ; no. 264, measles virus titre 1 : 4)
with 108 peptides. The sera with the higher titres reacted
with particular peptides loosely clustered in groups of
two or more peptides. This can partially be attributed to
sequences shared by adjacent peptides. Overall antibody
binding of serum no. 176 was more prominent than with
the low titre serum no. 264. However, only a few peptides
generated signals twofold above background. Both sera
6
II
16 21 26 31 36 4L 46 51 56 61 66 71 76 81 86 91 9 6 1 0 1 1 0 6
Fmoc-peptide
Fig. 2. Reactivity (mean of duplicate A,,os) of sera with a high (1:128)
(a) and a low (1:4) (b) anti-measles virus titre with 108 Fmoc
pentadecapeptides covering the whole sequence of the F protein.
markedly differed in the peptides which they recognized,
an observation extended in Fig. 3 to 30 unselected
donors, grouped by anti-measles virus titres. Binding was
very heterogeneous, but some sera showed similar
patterns. As shown in Fig. 3, binding patterns were
independent of the measles virus titres. Individual sera
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On: Sat, 17 Jun 2017 12:40:25
Measles virus F protein B cell epitopes
p.
I I I
I I I I I I I I I
I I I I I I
50
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60
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20
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16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 9 6 1 0 1 1 0 6
II
Peptide no.
Fig. 4. Percentage of sera reacting ( > t h r e e f o l d
individual peptides.
urn"
e
6
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background) with
I
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Fig. 5. Reactivity (multiple of background) with peptide no. 78 and
measles virus titres of the sera of Fig. 3.
t
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n
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Fig. 3. Antibody binding of 30 individual sera (grouped by antimeasles virus titres) to 108 F m o c - p e n t a d e c a p e p t i d e s of the F protein
(N, 2 to 2-99; W, 3 to 4.99; m, > 5 multiplicities of background).
Background was obtained for each serum by averaging the 54 lowest
values.
reacted with up to 24 peptides ( > threefold background;
one serum reacted with 32 peptides) or up to 44 peptides
( > twofold background). Fig. 4 shows the proportion of
sera that reacted with a given peptide (>threefold
background). Most peptides reacted with less than onethird of the sera. Peptides 6 and 78 reacted with more
than half of the sera. When the cutoff for a positive
reaction was lowered to twice the background, nine
peptides (peptides 6, 8, 26, 32, 39, 51, 78, 81 and 100)
reacted with more than 5 0 ~ of the sera (data not shown).
Sera with a titre of 1:128 appeared to bind to a few
peptides only. However, as for serum no. 176 (Fig. 2a)
this is partially due to the background defined by the
5 0 ~ lowest values.
Fig. 4 shows that the antibody-binding peptides are
loosely clustered in about eight to 10 groups covering
most but not all of the protein sequence. On the basis of
the cumulated data of Fig. 4, we have tentatively defined
nine regions containing B cell epitopes, covering
peptides 6 to 12, 24 to 32, 39 to 44, 48 to 54, 68 to 75, 75 to
82, 84 to 86, 90 to 95 and 99 to 104. Overlapping
sequences explain why most of the positive peptides were
within two peptides from other positive peptides.
However, the size of the clusters was larger than could be
explained by binding to sequences shared by adjacent
peptides.
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K.-H. Wiesmiiller and others
2214
I
1
I
l
i
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z~"--_~u~-~
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1111
I [I ~ b l
I .....
I i i 11 i i i i i i ~i 1115111
ill
, i ii i i i i I [ .......
ill
11 i i Ill
[~ .......
Fig. 6. B cell epitopes (I
, I I ......
11 , Ill
, ......
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I
,
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l
{
. . . .
I G I PAL I CCCRGRCNKKGEflVGMS
l
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I
l
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I
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I
[
. . . . .
I
. . . . . .
I
I
,
0 and acrophilicity, hydropathy, hydrophilicity plot.
The N-terminal ends of F1 (peptides 84 to 86) and F2
(peptides 101 to 104) are both reactive. About 25 ~ of the
sera reacted with the C-terminal peptide of F1. Mouse
antibodies to this peptide were found to react with the F
protein (Vialard et al., 1990). The strong reactivity with
peptides 6 and 7 may be due in part to covalent binding
to two cysteine residues. Peptides 8 to 11 and 81 to 86
correspond to putative transmembrane sequences. Peptides 50 and 51 correspond to a T cell epitope described
by Partidos & Steward (1990). This sequence also
induced antibodies in mice which, however, did not react
with the protein. Peptides 13 to 20, 33 to 38, 55 to 67, 87
to 89, 96 to 98 and 105 to 108 correspond to regions with
low antibody binding. Peptides 86 to 88 contain
sequences representing the C-terminal end of F2 and the
N-terminal end of F1. The peptides 105 to 108 represent
the signal sequence of the F protein and served as
negative controls.
We tested whether antibody-binding to the peptides
correlated with the anti-measles virus titres of the sera.
Fig. 5 shows this correlation for peptide 78. Up to a titre
of 1:64, maximal binding increased with serum titres.
Sera with a titre of 1 : 128 generated a lower binding due
to the background definition. The same analysis for the
above regions of enhanced binding gave a similar result:
maximal binding was mostly associated with antimeasles virus titres of 1 : 64 but not with sera with higher
titres.
Binding sites and structural features
Computer-assisted analysis of the sequence of the
measles virus F protein generated the probability for
each amino acid to participate in secondary structures
such as helices, fl-sheets, random coils and turns. No
obvious preferential binding to any one of the predicted
secondary structures became apparent (data not shown).
In addition, acrophilicity, the hydrophilicity and hydropathy patterns of the sequence were analysed according
to Kyte & Doolittle (1982) and Hopp & Woods (1981).
Fig. 6 schematically summarizes the latter structural
features and shows where the antibody binding sites are
located. The five C- and N-terminal amino acids of each
epitope have been omitted on the basis of the expected
reactivity with overlapping sequences. In summary this
comparison showed the following. (i) Antibody-binding
sites are preferentially but not exclusively located in
regions of transition of the most prominent hydropathic
with adjacent hydrophilic sequences. (ii) The hydropathic signal sequence is not reacting. (iii) Three
epitopes (peptides 39 to 44, 68 to 75, 90 to 95) do not
contain hydropathic sequences. (iv) The epitopes include
the putative transmembrane regions (amino acids 113 to
147 and 489 to 517). (v) The potential glycosylation sites
at amino acid positions 61 and 67 are probably not
reactive. (vi) The epitopes do not coincide with
acrophilic regions.
Discussion
Sera from unselected adult donors were used to map
antibody binding to linear epitopes of the measles virus F
protein. This approach identifies sequential B cell
epitopes both on the protein surface and in its core but
ignores conformational epitopes. The binding distribution varied markedly between individual donors and
was independent of the measles virus titre. Maximal
binding of a serum to a given peptide correlated with the
measles virus titre. Most sera recognized between 7 and
20~ of the overlapping peptides. The overall reactivity
was relatively weak but the cumulation of the binding
data from 30 donors defined eight to 10 clusters of
antibody binding, which we consider to be B cell
epitopes.
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Measles virus F protein B cell epitopes
These epitopes were located both on F1 and F2 and
were found near the N- and C-terminal ends of the two
subunits of the heterodimer. The epitopes included all of
the more hydropathic sequences at their transition to
adjacent hydrophilic sequences. The codons of the signal
sequence (amino acids 1 to 23) correspond to hydrophobic amino acids (1 to 20). Therefore the hydropathic
peptides corresponding to this sequence are adequate
negative controls.
Our approach does not discriminate between internal
and exposed B cell epitopes. The study shows that in late
convalescent donors, antibodies can be detected against
most peptides, indicating that both external sequences as
well as sequences embedded in the protein or membrane
core are reactive. Several other lines of evidence indicate
that this is the case. The B cell epitopes did not coincide
with acrophilic sequences. Putative transmembrane
regions (peptides 6 to 12 and 80 to 87) were recognized by
antibodies. The peptide 86 (KRFAGVVLAGAALGV)
represents the N terminus of F1 and includes the
transmembrane sequence FAGVVLAGAALGVATAAQIV which probably mediates cell fusion (Richardson
et al., 1980).
To analyse this further, we have used an algorithm
described by Parker & Hodges (1991) which predicts
more than 90% of surface sequences. The prediction is
based on the presence of tripeptides containing any of
the polar residues G, K, S, D, E, N, P, T, A, R and Q.
Peptides that do not contain polar tripeptide sequences
include peptides 7 to 9, 24, 30, 31, 50 to 53, 61 to 63, 67, 71
and 94 to 98 and are most probably inside the protein,
since the Parker and Hodges approach tends to
overestimate surface regions of proteins. According to
this prediction, the peptides 50 to 53 correspond to an
internal sequence of the F protein. Yet antibody
reactivity to these peptides is strong. Other putative
internal sequences showed little or no reactivity (peptides 30, 31, 61 to 63, 67, 71 and 96 to 98). In mice,
peptide 52 induced antibodies, but these were not
neutralizing [Partidos et al. (1991) and our own results].
Like this T cell epitope described by Partidos and
colleagues (Partidos & Steward, 1990; Partidos et al.,
1991), other hidden sequences are also unlikely to be
neutralizing. Whether these antibodies contribute to
immune protection, and which mechanisms may be
involved, remains unclear.
This also raises a question about the role of antibodies
recognizing linear sequences of the measles virus F
protein in protecting against reinfection. Although
antibodies to the F protein can neutralize the virus
(Malvoisin & Wild, 1990), it is not clear whether in the F
protein linear epitopes can be found which mediate virus
neutralization.
Pairing of B cell epitopes with T cell epitopes as found
2215
here in peptides 50 to 52 was also observed in other
pathogens such as influenza A virus (Barnett et al., 1989).
In influenza virus HA, B cell epitopes played a role in
defining T cell epitopes (Graham et al., 1989). Therefore,
antibody-binding studies with synthetic peptides may
not only be useful in identifying linear B cell epitopes but
may also help in the search for T cell epitopes.
Presumably, the antibodies detected here are transmitted passively to newborns (Albrecht et al., 1977). It is
possible to speculate, on the basis of the heterogeneity of
the inter-individual binding pattern, that it may be
possible to elicit an antibody response even in the
presence of measles virus antibodies by using synthetic
peptides. This could be of interest in inducing measles
virus antibodies in newborns despite persisting maternal
antibodies. Sequences spared by antibodies of natural
immunity are potential candidates for an antibodyresistant fractionated vaccine if such sequences can
induce neutralizing antibodies. In human immunodeficiency virus infections, it was shown that natural
immunity does not involve all possible immunogenic
peptides, leaving some capable of by-passing natural
antibodies (Rossi et al., 1989). Further studies however
are necessary to show, firstly, whether synthetic peptides
can elicit neutralizing antibodies against measles virus F
protein and, secondly, whether a peptide cocktail can be
designed which is (partially) resistant to (maternal) antimeasles virus antibodies.
Parts of this work were done by D.H. and G.S. in partial fulfilment of
their MD theses. D.H. and G.S. were supported by the Alphonse
Weicker Foundation, Luxembourg. We acknowledge the support of
the Centre de Recherche Publique-Sant+, of the Deutsche Krebshilfe,
and the valuable contribution of the Legs Kanning Foundation.
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2216
K.-H. Wiesmiiller and others
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(Received 18 February 1992; Accepted 4 May 1992)
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