D8586.PDF

Rev. sci. tech. Off. int. Epiz.,
1986, 5 (2), 411-427.
Monoclonal antibodies against morbilliviruses
1
K.C. McCULLOUGH* , H. SHESHBERADARAN**, E. NORRBY**, T. U. OBI*
and J.R. CROWTHER*
2
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 crossreactive 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 current 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 r u m i n a n t s ( P P R ) virus. M o n o ­
clonal antibodies (MAb) have been prepared against each of these isolates, and
their reactivity assayed by radio-immunoprecipitation assay ( R I P A ) , 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 t o identify epi­
topes on the virion proteins of measles, distemper and rinderpest viruses.
* Animal Virus Research Institute, Ash Road, Pirbright, Woking, Surrey G U 2 4 ONF, United
Kingdom.
** Dept. of Virology, Karolinska Institute, School of Medicine, c / o SBL, S-105 21 Stockholm,
Sweden.
1. Present Address: Ciba-Geigy A G , Centre de Recherches Agricoles, CH-1566 St-Aubin FR,
Switzerland.
2. Present Address: Department of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria.
!
— 412 —
I D E N T I F I C A T I O N OF V I R A L E P I T O P E S
A N D A N A L Y S I S OF M A b R E A C T I V I T I E S
M A b 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 M A b against homologous virus
are summarised in Table I (12, 14, 15). This work was extended to identify epitopes
o n rinderpest virus and to analyse heterologous reactivities of the anti-measles virus
and anti-distemper virus M A b (13, 16) as summarised in Tables II and III. Groupspecific (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 P P R and rinderpest viruses) epitopes were found
(16).
TABLE I
Epitope groups identified
by MAb against measles or distemper
virusa
Inducing
virusb
Virion protein
recognitionc
Number of epitope
groupsd
Measles virus
H
F
M
P
NP
10
6
6
3
5
H
F
M
P
NP
7
3
» »
Distemper virus
.e
6
18
a) F r o m N o r r b y el al. (12), S h e s h b e r a d a r a n el al. (15) and Orvell et al. (14).
b) T h e morbillivirus used for the immunising of B a l b / c mice from which splenocytes were s u b s e q u e n t l y o b t a i n e d for the
p r o d u c t i o n of h y b r i d o m a s .
c) T h e virion structural protein precipitated by the M A b .
d) T h e n u m b e r of M A b specificities identified by competition assays between M A b which precipitated the same virion
protein; this is not the complete set of specificities on each p r o t e i n , but the n u m b e r which were identifiable by the
M A b available.
e) N o anti-distemper virus M protein M A b were p r o d u c e d .
The anti-H protein M A b tended to be in the virus-specific category, although
some of the anti-measles virus H protein M A b did react with rinderpest viruses
(intertypic epitopes). In contrast, the epitopes on the F protein were mostly groupspecific. All but one of the anti-measles virus F protein M A b and majority of the
anti-distemper virus F protein M A b 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 M A b which were apparently identifying
a single epitope in the homologous competition assays (different M A b 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 M A b must have been detecting epitopes which were spatially too close to
be distinguishable by the homologous competition assays. F r o m this, a further sub­
division of epitope recognition by the M A b was obtained.
T A B L E II
Reactivity
MAba
1-29
1-41
B2-D
V17-D
1-44
9-DB10
10-EF10
19-DF10
81-53-D
16-AF10
A1-B2
R2-C3
16-AC5
16-EE9
of anti-measles
Virion protein
specificity
H
H
H
H
H
Fb
M
M
P
P
NP
NP
NP
NP
virus MAb
against rinderpest
virus
isolates
Rinderpest virus isolates used
RBOK
RBT/1
OMAN
YEMEN
+
+
+
+
+
-—
-—
+
+
+
—
+
-
-
-
-
+
+
+
+
+
+
+
+
-
—
—
-
-
-
+
+
+
+
-
-
-
-
+
+
+
+
—
-
+
+
+
-
-
-
+
-+
-
a) Examples of M A b which gave a particular p a t t e r n against rinderpest virus isolates.
b) All but o n e of the anti-measles virus F protein M A b gave this p a t t e r n of reactivity; the exception was 1 6 - A E 7 which
did not react with R B T / 1 or O M A N a n d only reacted with R B O K a n d Y E M E N in the i m m u n o f l u o r e s c e n c e test (16) a n d
ELISA.
T A B L E III
Reactivity
MAba
ALL
3.551
4.068
4.985
3.803
3.698
3.568
4.966
3.740
3.755
3.564
of anti-distemper
Virion protein
specificity
H
F
F
F
P
P
P
NP
NP
NP
NP
virus MAb
against rinderpest
virus
isolates
Rinderpest virus isolates used
RBOK
RBT/1
OMAN
YEMEN
—
•
_
+
_
-
+
•
—
+
-
-
+
+
—
—
+
-
•
•
—
+
-
-
+
+
+
-
•
—
—
-
+
+
—
-
a) Examples of M A b which gave a particular p a t t e r n of reactivity against rinderpest virus isolates.
The anti-M protein, anti-P protein and anti-NP protein M A b showed consider­
able variation in their reactivity against heterologous morbillivirus isolates (16)
(summarised in Tables II and III). Whilst the anti-M protein M A b reaction did not
further subdivide the epitope groupings, those against the P protein a n d N P protein
subdivided the distemper virus P protein epitope group 1 into three; the measles
virus N P protein epitope groups 2 and 3 into two and three respectively; and the
distemper virus N P protein epitope groups 6, 7 and 9 into two each, group 10 into
four and group 13 into three.
— 414 —
T A B L E IV
Epitope group recognition by the anti-morbillivirus
MAb:
subdivision of the groups based on reactivities with heterologous
virus
MAb
specificity
Reactivity against
isolates of
Original epitope
Virus
Measles
Protein
H
g
r o u
H
ALL
Measles
F
1,3,4
5,6
2
Distemper
Pa
Subdivision of
Measles
Distemper
Rinderpest
virus
virus
virus
la,6b,8
lb,2,7
3,4,5,6a
Distemper
+
+
+
+
+
+
+
+
+
+
+
±
2
3
M
P
1,3
2
Distemper
P
2,3,4,5,6
Measles
+
+
+
+
NP
None
None
None
la
lb
+
+
None
None
None
None
+
+
+
None
+
+
None
2a
2b
la
lb
lb
3a
3b
3c
(+
NP
None
None
None
None
None
+
Distemper
g
None
None
1
2,3
4,5
6
Measles
epitope
None
+
+
Measles
isolates
4,5
+
1,2,3,4,5
+
None
<+<•
+
i±<-
+
i
± c
+
(most isolates + )
!
±
c
\\±
c
+
+
6a
6b
7a
7b
+
(most isolates - )
±
+
±C
None
+
+
+
None
9a
9b
r o u
P
T A B L E IV (cont.)
MAb
specificity
Virus
Protein
Original epitope
groupa
Reactivity against
isolates of
Measles
virus
Distemper NP (cont.)
10
Distemper
virus
Rinderpest
virus
+
+
+
+
+
+
Subdivision of
epitope group
10a
10b
10c
10d
+
None
None
11
12
+
+
+
13
+
+
+
-
13a
13b
13c
14,15,16,17
+
—
None
±
±
c
a) The original e p i t o p e g r o u p referred to in T a b l e I a n d references 12, 14, 15.
b) Reactivity against all ( + ), s o m e ( ± ) or n o n e ( - ) of the isolates of this virus.
c) Different M A b in this g r o u p gave different p a t t e r n s of reactivity with the isolates of this virus — see T a b l e s II a n d III
and reference 16.
V A R I A T I O N OF M A b R E A C T I V I T I E S I N I M M U N O L O G I C A L 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 M A b reactivity in E L I S A , R I P A and I F (examples are shown in Table V). In
the former, all reactions against virion proteins may be detected; immunoprecipitation will only identify detergent-resistant epitopes reactive with " p r e c i p i t a t i n g "
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 E L I S A .
TABLE V
Differential
reactivity of anti-morbillivirus
MAb in ELISA,
immunoprecipitation
assay (RIPA) and immunofluorescence
test (IF)
Reactivity in
MAb
19-DC5
19-HF6
4.137
3.552
RPVc41
RPVc69
Inducing virus
Assay virus
ELISA
RIPA
IF
measles LEC
measles LEC
measles MANTOOTH
measles Hu2
+
+
-
+
distemper
distemper
rinderpest
rinderpest
distemper
distemper
rinderpest
rinderpest
+
+
+
+
-
+
+
CONVAC
CONVAC
RBOK
RBOK
ONDERSTEPOORT
ROCKBORN
RBOK
RBOK
-
•
-
-
— 416 —
Nevertheless, it is possible to identify epitopes on different morbillivirus iso­
lates, and to show how the different viruses are related. Greater information about
the degree of relatedness can be obtained by looking at the a m o u n t of M A b bound
to the virus. This can be calculated easily from E L I S A results, where the A
read­
ings are related to the a m o u n t of mouse immunoglobulin b o u n d in the test (Fig. 1).
The concentration of each virus sample used in the test is standardised through a
cross-reactive antibody which should give the same A
values against the same
concentration of each isolate. If the A
reading for this cross-reactive antibody
against a particular isolate — isolate A — is given the value of 100%, the A
read­
ings of the different M A b binding to isolate A can be expressed as a percentage
value relative to this.
4 9 2
4 9 2
4 9 2
4 9 2
5
10
µg/ml m o u s e
50
TOO
500
IgG
FIG. 1
Relationship of concentration of murine immunoglobulin ( µ g / m l 1) to the absorbance values at 492 nm ( A ) obtained from a sandwich ELISA in which rabbit IgG
anti-mouse immunoglobulin was absorbed to an ELISA plate, known concentra­
tions of murine immunoglobulin then reacted, and the quantity of b o u n d murine
immunoglobulin detected using peroxidase-conjugated rabbit anti-mouse immuno­
globulin. The bars give the standard deviation about each value.
4 9 2
Figure 2 shows an example of how the anti-rinderpest virus M A b can be used to
differentiate morbillivirus isolates by E L I S A . RPVc47 is a cross-reactive M A b ,
although it does bind more strongly to R P V isolates than to those of measles virus.
RPVc66 shows more discrimination, binding significantly more strongly to the
RBOK isolate of rinderpest virus than to other rinderpest virus isolates. This M A b
can also separate distemper virus isolates and a number of the measles viruses.
RPVc20 is a most discriminating M A b , reacting only with rinderpest RBOK virus
and measles M V O virus to any degree. The reactions of these anti-rinderpest virus
MAb can be summarised as in Table VI. Most of the M A b reacted against the M or
NP virion proteins. Some, like RPVc30, RPVc47, RPVc73 and RPVc75 reacted
with all of the virulent rinderpest virus isolates to a similar degree. (Interestingly,
some of these M A b did not react with any of the P P R virus isolates — this will be
dealt with in more detail below). Other M A b reacted only with the homologous
RBOK virus, the RBOK and O M A N isolates, or the RBOK, O M A N and R B T / 1
viruses. There was also an interesting number of M A b which did not react with the
homologous isolate (or with any rinderpest virus isolate in the case of RPVc22),
possibly because the epitopes recognised by the M A b were obscured in this particu­
lar type of E L I S A .
TABLE VI
Reactivity
MAba
c20
c24
c29
c30
c66
c73
c75
c47
c69
c42
c49
c68
c22
of anti-rinderpest
Virion protein
specificity*
virus MAb against rinderpest
RBT/1
M
M
M
M
NP
NP
NP
NP?x
+
+
+
+
+
+
_
?d
?e
isolates
Rinderpest virus isolate used
RBOK
?e
virus
OMAN
YEMEN
—
-
+
+
+
+
+
+
+
+
+
+
+
±
—
—
—
+
+
—
—
+
-
+
+
+
+
+
+
-
+
+
-
+
+
+
+
+
?f
(anti-measles virus)
a) Examples of MAb which gave a particular p a t l e r n of reactivity against rinderpest virus isolates.
b) Determined in R I P A using the h o m o l o g o u s RBOK isolate.
c) The R I P A did not give a clear indication of which protein precipitated, but the MAb a p p e a r e d to have N P specificity.
(I) This M A b did not react with RBOK in R I P A , but was positive by ELISA.
e) These M A b gave only h e t e r o l o g o u s reactions and then only by ELISA with rinderpest virus isolates.
f) This M A b reacted only with measles virus LEC-KI in E L I S A .
Similar results to those above with the anti-rinderpest virus M A b have been
found with anti-PPR virus M A b . Some of these antibodies reacted with all of the
rinderpest virus and P P R virus isolates, but to different degrees for each isolate
(Tables VII and VIII). A number of the anti-PPR virus M A b reacted selectively
with particular P P R virus isolates, although some of these — such as F 2 / C 3 — did
not differentiate the rinderpest virus isolates (Fig. 3). Another group of anti-PPR
virus M A b — exemplified by F 2 / F 7 in Fig. 3 — reacted more weakly with the P P R
— 418 —
FIG. 2
Analysis of the antigenic relationships of morbillivirus isolates using three antirinderpest virus (RBOK) M A b in an indirect ELISA. The A
values are expressed
as a percentage of a reference antiserum which cross-reacts with the morbilliviruses,
giving similar A
readings' for the same concentrations of each virus in this indi­
rect ELISA. The measles virus (MV), distemper virus (CDV) a n d rinderpest virus
(RPV) isolates are as detailed in Sheshberadaran et al. (16).
4 9 2
4 9 2
— 419 —
than with the rinderpest virus isolates. A n u m b e r of a n t i - P P R virus M A b reacted
with all P P R virus isolates but n o t with any rinderpest virus. U n f o r t u n a t e l y , the
hybridomas were lost t h r o u g h u n f a v o u r a b l e e n v i r o n m e n t a l changes in t h e hybridoma unit.
TABLE V I I
Reactivity
of anti-PPR
virus MAb
against rinderpest
virus
isolates
Rinderpest virus isolates used
MAba
F1/C6
F1/C3
F1/B12
F1/E8
F1/H8
RBOK
RBT/1
OMAN
YEMEN
LEBANON
EGYPT
+ + +
(0.91)b
+ + +
(1.06)
+ + +
(0.80)
+ + +
(0.91)
+ +
(0.65)
+ + +
(0.82)
+ + +
(1.21)
+ + +
(0.91)
+ + +
(0.94)
+ + +
(1.09)
+ + +
(0.86)
+ + +
(1.20)
+ + +
(0.92)
+
(0.37)
+ + +
(1.21)
+ + +
(1.01)
+ + +
(1.38)
+ + +
(1.02)
+ + +
(1.04)
+ + +
(1.14)
+ + +
(0.85)
+ +
(0.63)
+
(0.36)
+
(0.42)
+
(0.39)
+ + +
(0.90)
+ +
(0.77)
+
(0.51)
+ + +
(0.98)
+
(0.41)
•
a} Examples of M A b which gave a particular p a t t e r n of reactivity against rinderpest virus isolates.
b) The actual A492 values o b t a i n e d .
TABLE VIII
Reactivity
of anti-PPR
virus MAb
against
PPR virus
isolates
P P R virus isolates used
MAba
F1/A5
F1/B3
F1/B4
F1/B6
F1/B7
F1/E8
Fl/Fl 1
F1/G12
F1/G5
F1/H7
75/1
75/2
75/3
76/1
ACCRA
+ + +
(0.89)£
+
(0.44)
+
(0.36)
+
(0.35)
+
(0.39)
+ +
(0.66)
+ + +
(0.89)
+ + +
(0.96)
+ + +
(0.86)
+ + +
(0.83)
+ + +
(0.75)
+
(0.53)
+
(0.40)
+
(0.30)
+
(0.42)
+ +
(0.62)
+ + +
(0.94)
+
(0.41)
+
(0.30)
+
(0.40)
+ + +
(0.86)
+ + +
(0.96)
+ + +
(0.95)
+ + +
(0.91)
+ + +
(1.03)
+ +
(0.70)
+ + +
(0.90)
+ +
(0.70)
+ + +
(0.76)
+ + +
(0.78)
+ + +
(0.89)
+ +
(0.70)
+
(0.39)
+ +
(0.65)
+ +
(0.66)
+
(0.40)
+ + +
(0.99)
+ +
(0.67)
+ + +
(0.82)
+ +
(0.59)
+ + +
(0.91)
+ + +
(0.93)
+ +
(0.69)
+ +
(0.71)
+
(0.40)
+
(0.35)
+
(0.32)
+ + +
(0.91)
+ + +
(0.84)
+ + +
(0.88)
") Examples of M A b which gave a particular p a t t e r n of reactivity against P P R virus isolates.
b) The actual A
values o b t a i n e d .
492
— 420 —
FIG. 3
Examples of the reactivity patterns of anti-PPR virus M A b
rinderpest virus isolates in the indirect ELISA, using the
F 2 / C 3 , and F 2 / F 7 . The P P R viruses were 7 5 / 1 , 7 5 / 2 , 7 5 / 3
pest virus isolates were RBOK, Lebanon, Egypt
against P P R virus and
MAb F1/D4, F2/A1,
and Accra; the rinder­
and O m a n .
— 421 —
Where a M A b cannot react with a particular heterologous virus, the antigenic
site which would correspond to that with which the M A b binds on the homologous
virus must have been considerably altered. Those M A b which show a reduction in
activity for a heterologous virus, under standardised conditions (to account for
fluctuations in concentrations of different virus preparations), are detecting an epi­
tope which also must have been modified, but to a lesser degree than that which
gives no reaction with the M A b . Since the antibodies are monoclonal, it is likely
that this reduction is reflective of an alteration in the binding capacity (affinity?) of
the MAb which, in turn, would suggest alterations in the antigenic determinant so
that some of the sites for bonding with the antibody p a r a t o p e are lost from the
altered epitope.
Thus, from E L I S A titrations of M A b against different virus isolates, it is pos­
sible to give each isolate an "antigenic fingerprint". In this way, a particular test
isolate can be related to known viruses. This would assist with the epidemiological
investigations of the diseases involved, but could also be a useful guide to the p r o ­
tective capacity of a vaccine against a field virus. The latter work would use M A b
which were related to the protective immune response against the virulent form of
the vaccine virus (that is, they efficiently opsonise virus, thus enhancing phagocyto­
sis — see reference 9; M A b which neutralise virus infectivity may be opsonins, but
neutralisation of the infectivity per se is not the complete picture and m a y be mis­
leading — see reference 11). If such M A b which identified protection-associated
epitopes on the vaccine virus also b o u n d t o a field isolate, then a vaccinated animal
should have a high probability of being protected. The greater the antigenic
relationship at the protection-associated epitopes between field and vaccine virus,
the greater would be the confidence in the protective capacity of that vaccine.
However, these assays would give no information on the i m m u n e response genera­
ted by the vaccine, then the animals should be protected.
Consequently, a second test must be employed. This is a competition assay in
which vaccinated animal antisera are tested for their capacity to block the binding
of protective M A b to vaccine virus or field isolate. Such work has been done with
MAb against foot-and-mouth disease virus (2), and shown to be a valid test system.
An opsonisation assay such as the liquid-phase E L I S A (10) is the most appropriate
(see references 9, 11), and to this end Hamblin et al. (4, 5) have developed a liquidphase blocking E L I S A which directly relates particular levels of opsonising anti­
body (anti-FMD virus) to protection.
We are currently adapting our anti-morbillivirus M A b to competition assays for
the diagnosis of rinderpest and P P R immunity, and the diagnosis and identification
of rinderpest and P P R virus isolates. In this latter situation, we have three antirinderpest virus M A b which can distinguish rinderpest virus isolates from P P R
virus isolates. One of these — RPVc75 — is shown in Fig. 4 alongside the crossreactive RPVc47. N o n e of the P P R virus isolates (and this has now been extended
to include all P P R viruses currently available) b o u n d RPVc75, whereas all rinder­
pest virus isolates did. H a d conditions been more favourable, a n t i - P P R virus M A b ,
which distinguished P P R virus isolates from those of rinderpest virus, would have
survived. These were obtained from only one out of eight fusions, but attempts are
being made to generate such M A b once again, using separated virion proteins.
An interesting observation with the M A b such as RPVc75 was that the Egypt
isolate of rinderpest virus reacted very weakly (Fig. 4). This virus shows greatly
reduced virulence in vivo compared to the other isolates, producing mild symptoms
— 422 —
FIG. 4
Differentiation of rinderpest virus from P P R virus using anti-rinderpest virus MAb
in the indirect ELISA. The upper plot shows the reactivities with the rinderpest
virus isolates; the lower plot shows the reactivities with the P P R virus isolates. The
M A b used are the cross-reactive RPVc47 ( • ),. which will show the relative
amounts of virion antigens which have been adsorbed to the E L I S A plate, and one
of the differentiating M A b — RPVc75 ( • ).
— 423 —
in susceptible cattle; it is also incapable of totally destroying a cultivated cell m o n o ­
layer. It would be interesting to look at the Tanzanian isolate, which is in a similar
category to this rinderpest Egypt virus, to see if M A b could identify virulencerelated epitopes on rinderpest virus.
In summary, using M A b against measles, distemper, rinderpest and P P R
viruses, we have demonstrated an antigenic relationship between the four major
morbilliviruses; the virus proteins are related, not just physico-chemically, but also
antigenically. The only exception to this is the distemper virus H protein, on which
we can find no epitope shared with other morbilliviruses. F r o m the degree of anti­
genic cross-reaction between the proteins of the morbilliviruses, we have proposed
an evolutionary tree for the group (12). This has now been extended as shown in
Fig. 5, where P P R virus appears to be a relatively recent evolution from the
FIG. 5
Morbillivirus evolution
Proposed evolutionary tree for the morbillivirus group as determined from the reactivities of
the MAb against heterologous morbillivirus types — elaborated from Norrby et al. (13). The
figure arbitrarily indicates the antigenic variations in the viruses by the arrows, and shows the
relative positions on the evolutionary progress of the group at which distemper virus (CDV),
measles virus (MV) and PPR virus (PPRV) may have evolved from the archetype rinderpest
virus (RPV). N o indication of the exact time scale or the number of antigenic changes has
been, or can be, given.
— 424 —
archetype rinderpest virus — in fact the P P R virus may only be a host range variant
of rinderpest virus (although measles and distemper viruses could be similarly
described). The recent evolution of P P R viruses should enable us to mimic this
adaptation to small ruminants; the relatively avirulent Egypt and Tanzania isolates
of rinderpest virus may prove useful in this context, since they appear to be
somewhere between the " t r u e " rinderpest virus which is highly pathogenic for cer­
tain breeds of cattle, and the " t r u e " P P R virus which is asymptomatic in bovine
species. These " m i l d " strains of rinderpest virus (Egypt and Tanzania) may also
produce a latent rinderpest infection. Such a relationship between reduced patho­
genicity and latency has been demonstrated with measles virus (8, 17). The
influence which " m i l d " a n d / o r latent rinderpest viruses would have on the epizootiology of the disease is certainly of concern, particularly after a vaccination pro­
g r a m m e has been discontinued due to the apparent absence of rinderpest in a parti­
cular area.
This topic of host-pathogen interactions in morbillivirus infections is currently
being studied with the aid of M A b , particularly with respect to the " i m m u n o s u p ­
p r e s s i o n " associated with the diseases. These antibodies, and the M A b against
leukocyte markers, can identify the development of virion antigens in subpopula­
tions of infected leukocytes. From this, it should be possible to identify differences
between the virulent and " m i l d ' / ' a v i r u l e n t " viruses in the proteins produced on
the cell surface, and the degree of virus-induced cell damage. As an extension, the
antibody-dependent and antibody-independent cytotoxicity reaction can be studied
to see how the lymphopenia associated with the in vivo infections by these viruses is
affected. With measles virus, the virus preferentially infects the O K T
helper/inducer lymphocyte population (7), but only produces a substantial lympho­
penia in vivo (1). Nevertheless, the virus can directly disrupt the proliferative capa­
city and function of different lymphocyte subsets in vivo (1, 3, 6, 18); it was interes­
ting that separated virion proteins of measles virus could also impair the prolifera­
tive response of lymphocytes (18). Despite these effects of the virus, both a protec­
tive i m m u n e response and an effective immunological m e m o r y develop.
Furthermore, immune lymphocytes are apparently less susceptible to the "immunod e p r e s s a n t " effects of morbilliviruses (3). With the M A b available against morbilli­
virus proteins and both bovine and ovine leukocyte markers, it should be possible
to identify at least some of the processes involved in the h o s t / p a t h o g e n interactions
in rinderpest and P P R virus infections. Since these can also be studied relatively
easily in the host animal, the work can be extrapolated to provide information
about the situation with measles in h u m a n s .
+
4
ACKNOWLEDGEMENTS
We wish to thank Dr W . P . Taylor (AVRI) for supplying the original stocks of
the rinderpest and P P R viruses, Dr C. Orvell (Karolinska Institute) for the antidistemper virus M A b , Drs D. McFarlin and K.W. R a m m o h a n for the antiEdmonston isolate of measles virus M A b , and Robin Butcher, Bill Carpenter,
A n n a Coter and Mariethe Ehnlund for their excellent technical contributions to the
experimental work quoted in this article.
*
**
— 425
ANTICORPS MONOCLONAUX CONTRE LES MORBILLIVIURS. — K.C. McCullough, H. Sheshberadaran, E. Norrby, T.U. Obi et J.R. Crowther.
Résumé : Les anticorps monoclonaux contre les virus de la rougeole, de la
maladie de Carré et de la peste bovine ont été utilisés pour mettre en évidence
la parenté et les différences antigéniques entre les quatre morbillivirus (ceux de
la rougeole, de la maladie de Carré, de la peste bovine et de la peste des petits
ruminants). Les résultats ont montré que les protéines constitutives des virions
de ces morbillivirus étaient apparentées entre elles du point de vue antigénique
comme du point de vue physico-chimique, à l'exception de la protéine H du
virus de la maladie de Carré, dont aucun des épitopes détectés par les anticorps monoclonaux n'était partagé avec ceux des protéines H des autres virus.
C'est la protéine F des virus qui a présenté le degré le plus élevé d'homologie
épitopique entre les types de morbillivirus, tandis que les épitopes de la protéine H étaient surtout spécifiques de type. Les protéines internes des virions
— M, P et NP — portaient des épitopes à la fois spécifiques de type et à réaction croisée. La plupart des épitopes avaient été identifiés au moyen d'épreuves de compétition; mais les différences de réactivité de certains anticorps
monoclonaux vis-à-vis des différentes souches étudiées ont montré que les
anticorps, dont on pensait précédemment qu'ils reconnaissaient des épitopes
identiques, réagissaient probablement avec des épitopes très rapprochés dans
l'espace. A partir de ce travail, il a été possible de différencier les morbillivirus
avec précision et d'étendre cette méthode à la séparation des souches de virus
isolées, en assignant à chaque souche de morbillivirus une "empreinte antigénique" particulière. Ainsi, des analyses épidémiologiques précises peuvent être
réalisées, et une technique ELISA, particulièrement utile pour différencier
rapidement et exactement les virus de la peste bovine des virus de la peste des
petits ruminants, a été mise au point. La discussion de cette étude porte sur
l'application actuelle des anticorps monoclonaux à l'identification des mécanismes de protection immunitaire humorale, les épitopes des virions qui s'y
rapportent, et l'aptitude des morbillivirus à attaquer les défenses de l'hôte en
produisant un effet immunodépresseur caractéristique.
MOTS-CLÉS : Anticorps monoclonaux - Diagnostic différentiel - Epidémiologie - Immunologie - Morbillivirus - Structure antigénique - Virus de la maladie de Carré - Virus de la peste bovine - Virus de la peste des petits ruminants Virus de la rougeole.
*
ANTICUERPOS MONOCLONALES CONTRA LOS MORBILIVIRUS. — K.C. McCullough, H. Sheshberadaran, E. Norrby, T.U. Obi y J.R. Crowther.
Resumen : Se han utilizado los anticuerpos monoclonales contra los virus del
sarampión, enfermedad de Carré y peste bovina para evidenciar el parentesco
y las diferencias antigénicas entre los cuatro morbilivirus (los del sarampión,
enfermedad de Carré, peste bovina y peste de pequeños rumiantes). Los resultados demostraron que las proteínas constitutivas de los viriones de estos morbilivirus estaban emparentadas entre sí, tanto antigénica como fisicoquímicamente, con excepción de la proteína H del virus de la enfermedad de Carré, de
la cual ninguno de los epitopos detectados por los anticuerpos monoclonales
estaba compartido con los de las proteínas H de los demás virus. La proteína
F de los virus es la que presentó el más elevado grado de homología epitópica
entre los tipos de morbilivirus, mientras que los epitopos de la proteína H eran
sobre todo específicos de tipo. Las proteínas internas de los viriones — M, Py
— 426 —
NP — tenían epitopos a la vez específicos de tipo y de reacción cruzada. Se
habían identificado la mayor parte de los epitopos mediante pruebas de competición; pero las diferencias de reactividad de algunos anticuerpos monoclonales frente a las distintas cepas estudiadas demostraron que los anticuerpos,
de los que se pensaba anteriormente que reconocían epitopos idénticos, probablemente reaccionaban con epitopos muy próximos en el espacio. En base a
este trabajo, se pudieron diferenciar los morbilivirus con precisión y extender
el método a la separación de las cepas de virus aisladas, asignando a cada cepa
de morbilivirus una "huella antigénica" peculiar. Se pueden así realizar análisis epidemiológicos precisos, habiéndose elaborado una técnica ELISA, de
suma utilidad para diferenciar con celeridad y exactitud los virus de la peste
bovina de los virus de la peste de pequeños rumiantes. La discusión de este
estudio se refiere a la actual aplicación de los anticuerpos monoclonales para
la identificación de los mecanismos de protección inmunitaria humoral, los
epitopos de los viriones que se refieren a la misma, y la aptitud de los morbilivirus para atacar las defensas del huésped produciendo un efecto inmunodepresor característico.
PALABRAS CLAVE : Anticuerpos monoclonales - Diagnóstico diferencial Epidemiología - Estructura antigénica - Inmunología - Morbillivirus - Virus de
la enfermedad de Carré - Virus de la peste bovina - Virus de la peste de
pequeños rumiantes - Virus del sarampión.
REFERENCES
1. CASALI P . , RICE G . P . A . & OLDSTONE M . B . A . (1984). — Viruses disrupt functions of
human lymphocytes. Effects of measles virus and influenza virus on lymphocytemediated killing and antibody production. J. Exp. Med., 159, 1322-1337.
2. CROWTHER J . R . , MCCULLOUGH K.C., BROCCHI E. & D E SIMONE F . (1984). — Monoclo-
nal antibodies against foot-and-mouth disease virus: application and potential use. Eur.
Comm. Cont. FMD, Brescia, Italy, June 1984, published by FAO, Rome.
3. GALAMA J . M . D . , UBELS-POSTMA J . , Vos A. & LUCAS C . J . (1980). — Measles virus
inhibits acquisition of lymphocyte functions but not established effector functions. Cell.
Immunol., 50, 405-415.
4. HAMGLIN C , BARNETT I. & HEDGER R. (1986). — A new enzyme-linked immunosorbent
assay (ELISA) for the detection of antibodies against foot-and-mouth disease virus. I.
Development and method of ELISA. J. Meth. Immunol. (in press).
5. HAMBLIN C , BARNETT I. & CROWTHER J . R . (1986). — A new enzyme-linked immuno-
sorbent assay (ELISA) for the detection of antibodies against foot-and-mouth disease
virus. II. Application. J. Meth. Immunol. (in press).
6. ILONEN J . , LANNING M . , HERVA E. & SALMI A. (1980). — Lymphocyte blast transfor-
mation responses in measles infection. Scand. J. Immunol.,
12, 383-391.
7. JACOBSON S. & MCFARLAND H . F . (1984). — Measles virus infection of human peripheral
blood lymphocytes; importance of the O K T 4 T-cell subset. In: D . H . Bishop and R.W.
Compans, Nonsegmented Negative Strand Viruses, Alabama, U.S.A., 1983, published
by Academic Press, New-York, 435-442.
+
8. MCCULLOUGH K.C. (1983). — Characterisation of a non-syncytiogenic autonomously
replicating variant of measles virus. J. gen. Virol., 64, 749-754.
— 427 —
9. MCCULLOUGH K . C . & CROWTHER J . R . (1985). — The protective immune response
against foot-and-mouth disease virus: relationship to virion topography.
Mouth Disease Bulletin, 23, 8, 1-4.
Foot-and-
10. MCCULLOUGH K . C . , CROWTHER J . R . & BUTCHER R . N . ( 1 9 8 5 ) . — A liquid-phase ELISA
and its use in the identification of epitopes on foot-and-mouth disease virus antigens. J.
Virol. Methods, 11, 329-338.
11. MCCULLOUGH K . C . , CROWTHER J . R . , BUTCHER R . N . , CARPENTER W . C . , BROCCHI E.,
CAPUCCI L. & D E SIMONE F. (1986). — Immune protection against foot-and-mouth
disease virus studied using virus neutralising and non-neutralising concentrations of
monoclonal antibodies. Immunol. (in press).
12. NORRBY E., CHEN S . - N . , TOGASHI T., SHESHBERADARAN H . & JOHNSON K . P . ( 1 9 8 2 ) . —
Five measles virus antigens demonstrated by use of mouse hybridoma antibodies in pro­
ductively infected tissue culture cells. Arch. Virol., 71, 1-11.
13. NORRBY E., SHESHBERADARAN H . , MCCULLOUGH K . C . , CARPENTER W . C . & ORVELL C.
(1985). — Is rinderpest virus the archevirus of the morbillivirus genus? Intervirology, 23,
228-232.
14. ORVELL C . , SHESHBERADARAN H . & NORRBY E. ( 1 9 8 5 ) . — Preparation and characterisa­
tion of monoclonal antibodies directed against four structural components of canine dis­
temper virus. J. gen. Virol., 66, 443-456.
15. SHESHBERADARAN H . , CHEN S . - N . & NORRBY E. ( 1 9 8 3 ) . — Monoclonal antibodies
against five structural components of measles virus. I. Characterisation of antigenic
determinants on nine strains of measles virus. Virology, 128, 341-353.
16. SHESHBERADARAN H . , NORRBY E., MCCULLOUGH K . C . , CARPENTER W . C . & ORVELL C.
(1986). — The antigenic relationship between measles, canine distemper and rinderpest
viruses studied with monoclonal antibodies. J. gen. Virol. (in press).
17. THORMAR H . , MEHTA P . D . & BROWN H . R . ( 1 9 7 8 ) . — Comparison of wild-type and
subacute sclerosing panencephalitis strains of measles virus. J. Exp. Med., 148, 6 4 7 - 6 9 1 .
18. ZWEIMAN B . , LISAK R . P . , WATERS D . & KOPROWSKI H . ( 1 9 7 9 ) . — Effects of purified
measles virus components on proliferating human lymphocytes. Cell. Immunol., 47, 2 4 1 247.