D8586.PDF

Rev. sci. tech. Off. int. Epiz., 1986, 5 (2), 411-427.
Monoclonal antibodies against morbilliviruses
K.C. McCULLOUGH*1, H. SHESHBERADARAN**, E. NORRBY**, T. U. OBI*2
and J.R. CROWTHER*
Summary: Monoclonal antibodies (MAb) against measles virus, distemper
virus and rinderpest virus were used to demonstrate antigenic relationship and
differences between all four morbilliviruses (measles, distemper, rinderpest
and peste des petits ruminants viruses). From this, it was shown that the con-
stituent virion proteins of these morbilliviruses were related antigenically as
well as physico-chemically, with the exception of the distemper virus H pro-
tein on which none of the epitopes detected by the monoclonal antibodies
were shared with the H proteins of the other viruses. The F protein of the\
viruses showed the greatest degree of epitope homology between morbillivirus
types, while the H protein epitopes were mostly type-specific. The internal
virion proteins — M, P and NP — carried both type-specific and cross-
reactive epitopes. Most of the epitopes had been identified using competition
assays; but differences in the reactivity of certain MAb against different isola-
tes showed that antibodies previously thought to be recognising identical epi-
topes were probably reacting with spatially very close epitopes. From this
work, it has been possible to accurately differentiate the morbilliviruses, and
to extend this to the separation of isolates through assigning particular "anti-
genic fingerprints" to each morbillivirus isolate. Thus accurate epidemiologi-
cal analyses can be performed, and a particularly useful ELISA for the rapid
and accurate differentiation of rinderpest viruses from peste des petits rumi-
nants viruses has been developed. The work is discussed in terms of the cur-
rent application of the MAb to the identification of protective humoral
immune mechanisms, virion epitopes of relevance to this, and the capacity of
morbilliviruses to attack the host defences, producing the characteristic
depression of immune responsiveness.
KEYWORDS: Antigenic structure - Differential diagnosis - Distemper virus -
Epidemiology - Immunology - Measles virus - Monoclonal antibodies - Mor-
billiviruses - Peste des petits ruminants virus - Rinderpest virus.
The morbillivirus group consists of four major virus members — measles virus,
distemper virus, rinderpest virus and peste des petits ruminants (PPR) virus. Mono-
clonal antibodies (MAb) have been prepared against each of these isolates, and
their reactivity assayed by radio-immunoprecipitation assay (RIPA), immunofluo-
rescence (IF) and enzyme-linked immunosorbent assay (ELISA) (12, 14, 15;
McCullough et al., in preparation; Obi and McCullough, in preparation). The
MAb which precipitated individual virion polypeptides were used to identify epi-
topes on the virion proteins of measles, distemper and rinderpest viruses.
* Animal Virus Research Institute, Ash Road, Pirbright, Woking, Surrey GU24 ONF, United
Kingdom.
** Dept. of Virology, Karolinska Institute, School of Medicine, c/o SBL, S-105 21 Stockholm,
Sweden.
1. Present Address: Ciba-Geigy AG, Centre de Recherches Agricoles, CH-1566 St-Aubin FR,
Switzerland.
2. Present Address: Department of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria.
!

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IDENTIFICATION OF VIRAL EPITOPES
AND ANALYSIS OF MAb REACTIVITIES
MAb with the same polypeptide specificity were used in competition assays
against whole virus to determine the number of detectable epitopes. The results
with the anti-measles virus and anti-distemper virus MAb against homologous virus
are summarised in Table I (12, 14, 15). This work was extended to identify epitopes
on rinderpest virus and to analyse heterologous reactivities of the anti-measles virus
and anti-distemper virus MAb (13, 16) as summarised in Tables II and III. Group-
specific (on all morbilliviruses), group cross-reactive (on some, but not all, isolates
of measles, distemper and rinderpest viruses), type-specific (unique to measles, dis-
temper or rinderpest virus only) and intertypic (shared between at least some of the
isolates of two of the morbillivirus types: either measles and rinderpest viruses, dis-
temper and rinderpest viruses or PPR and rinderpest viruses) epitopes were found
(16).
TABLE I
Epitope groups identified by MAb against measles or distemper virusa
Inducing Virion protein
Number of epitope
virusb recognitionc groupsd
Measles virus
H 10
F 6
» » M 6
P 3
NP 5
Distemper virus
H 7
F 3
M .e
P 6
NP 18
a) From Norrby el al. (12), Sheshberadaran el al. (15) and Orvell et al. (14).
b) The morbillivirus used for the immunising of Balb/c mice from which splenocytes were subsequently obtained for the
production of hybridomas.
c) The virion structural protein precipitated by the MAb.
d) The number of MAb specificities identified by competition assays between MAb which precipitated the same virion
protein; this is not the complete set of specificities on each protein, but the number which were identifiable by the
MAb available.
e) No anti-distemper virus M protein MAb were produced.
The anti-H protein MAb tended to be in the virus-specific category, although
some of the anti-measles virus H protein MAb did react with rinderpest viruses
(intertypic epitopes). In contrast, the epitopes on the F protein were mostly group-
specific. All but one of the anti-measles virus F protein MAb and majority of the
anti-distemper virus F protein MAb reacted with other morbilliviruses. There was a
lower level of epitope sharing between distemper and measles viruses, and between
distemper and rinderpest viruses, than between measles and rinderpest viruses.
Some of the anti-distemper virus F protein MAb which were apparently identifying
a single epitope in the homologous competition assays (different MAb competing
for binding to the homologous/inducing virus) had a differential reactivity against
heterologous virus isolates. Examples of this are shown in Table IV, and suggest
that these MAb must have been detecting epitopes which were spatially too close to
be distinguishable by the homologous competition assays. From this, a further sub-
division of epitope recognition by the MAb was obtained.

TABLE II
Reactivity of anti-measles virus MAb against rinderpest virus isolates
MAba
Virion protein
specificity
Rinderpest virus isolates used
MAba
Virion protein
specificity RBOK RBT/1 OMAN YEMEN
1-29 H + + + +
1-41 H + + + —
B2-D H - - + —
V17-D H — — — +
1-44 H - - - -
9-DB10
Fb + + + +
10-EF10
M + + + +
19-DF10 M - - - -
81-53-D P + + + +
16-AF10
P -- - -
A1-B2 NP + + + +
R2-C3 NP -+ + +
16-AC5
NP + — -+
16-EE9 NP - - - -
a) Examples of MAb which gave a particular pattern against rinderpest virus isolates.
b) All but one of the anti-measles virus F protein MAb gave this pattern of reactivity; the exception was 16-AE7 which
did not react with RBT/1 or OMAN and only reacted with RBOK and YEMEN in the immunofluorescence test (16) and
ELISA.
TABLE III
Reactivity of anti-distemper virus MAb against rinderpest virus isolates
MAba
Virion protein
specificity
Rinderpest virus isolates used
MAba
Virion protein
specificity RBOK RBT/1 OMAN YEMEN
ALL H —
3.551 F • + • •
4.068
F _ _ — +
4.985
F -- -
3.803
P + • + +
3.698
P — + +
3.568
P - - -
4.966
NP + + • +
3.740
NP — — — +
3.755
NP + — —
3.564
NP -- -
a) Examples of MAb which gave a particular pattern of reactivity against rinderpest virus isolates.
The anti-M protein, anti-P protein and anti-NP protein MAb showed consider-
able variation in their reactivity against heterologous morbillivirus isolates (16)
(summarised in Tables II and III). Whilst the anti-M protein MAb reaction did not
further subdivide the epitope groupings, those against the P protein and NP protein
subdivided the distemper virus P protein epitope group 1 into three; the measles
virus NP protein epitope groups 2 and 3 into two and three respectively; and the
distemper virus NP protein epitope groups 6, 7 and 9 into two each, group 10 into
four and group 13 into three.

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TABLE IV
Epitope group recognition by the anti-morbillivirus MAb:
subdivision of the groups based on reactivities with heterologous virus isolates
MAb Reactivity against
specificity isolates of
Original epitope Subdivision of
Virus Protein grouPa Measles Distemper Rinderpest epitope grouP
virus virus virus
Measles
Distemper
Measles
Distemper
Measles
Measles
Distemper
Measles
H
H
F
M
P
P
NP
Distemper NP
la,6b,8
lb,2,7
3,4,5,6a
ALL
1,3,4
5,6
2
2
3
1
2,3
4,5
6
1,3
2
2,3,4,5,6
4,5
1,2,3,4,5
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
(+
+
+ +
<+<• +
i±<- +
i±c +
(most isolates +)
!±c
+
(most isolates - )
± +
\±C
+
\±c +
+
+
+
+
±
+
+
+
+
None
None
None
None
None
None
None
None
None
la
lb
None
None
None
None
None
None
None
la
lb
lb
None
2a
2b
3a
3b
3c
None
None
6a
6b
7a
7b
None
9a
9b

TABLE IV (cont.)
MAb
specificity
Virus Protein
Original epitope
groupa
Reactivity against
isolates of
Measles Distemper Rinderpest
virus virus virus
Subdivision of
epitope group
Distemper NP (cont.)
10
11
12
13
14,15,16,17
+ + 10a
+ + 10b
+ 10c
+ + 10d
+ + None
+ ± None
+ 13a
+
±c
13b
+ -13c
+ — None
a) The original epitope group referred to in Table I and references 12, 14, 15.
b) Reactivity against all ( + ), some ( ± ) or none ( - ) of the isolates of this virus.
c) Different MAb in this group gave different patterns of reactivity with the isolates of this virus — see Tables II and III
and reference 16.
VARIATION OF MAb REACTIVITIES IN IMMUNOLOGICAL TESTS
These experiments highlighted the difficulty in accurately identifying all epi-
topes by a single assay. The differentiation can be further complicated by compa-
ring MAb reactivity in ELISA, RIPA and IF (examples are shown in Table V). In
the former, all reactions against virion proteins may be detected; immunoprecipita-
tion will only identify detergent-resistant epitopes reactive with "precipitating"
murine antibodies; the IF test identifies reactivities against infected cells. Not all
MAb will be usable in all of the tests; for example, the two anti-rinderpest virus
MAb shown in Table V cannot be characterised in terms of which virion proteins
they react with, because they are detected only by ELISA.
TABLE V
Differential reactivity of anti-morbillivirus MAb in ELISA, immunoprecipitation
assay (RIPA) and immunofluorescence test (IF)
Reactivity in
MAb Inducing virus Assay virus ELISA RIPA IF
19-DC5 measles LEC measles MANTOOTH + -•
19-HF6 measles LEC measles Hu2 + -+
4.137 distemper CONVAC distemper ONDERSTEPOORT + -+
3.552 distemper CONVAC distemper ROCKBORN + -+
RPVc41 rinderpest RBOK rinderpest RBOK + -
RPVc69 rinderpest RBOK rinderpest RBOK + -