Journal of General Virology (1991), 72, 549-555.
Printed in Great Britain
549
Antigenic relatedness between arenaviruses defined at the epitope level by monoclonal antibodies S. L. Ruo,* S. W. Mitchell, M. P. Kiley, L. F. Roumillat, S. P. Fisher-Hoch and J. B. McCormick Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, Georgia 30333, U.S.A.
Monoclonal antibodies (MAbs) were produced against two African arenaviruses, Lassa virus and Mopeia virus. Competitive binding analysis of MAbs identified four antigenic sites on the nucleoprotein (NP), two on glycoprotein 1 (GP1) and six on glycoprotein 2 (GP2) of the Josiah strain of Lassa virus. 64 virus isolates from western, central and southern Africa were all consistently distinguishable by MAbs to certain epitopic sites on GP1, GP2 and NP viral proteins.
Furthermore, MAbs to Lassa virus GP1 and NP uniformly distinguished viruses from the West African countries of Sierra Leone, Liberia and Guinea from those of Nigeria. GP2-directed MAbs to two African arenaviruses reacted broadly with South American arenaviruses demonstrating that an epitopic site on GP2 may be the most highly conserved antigen in the arenavirus group.
Introduction
tural proteins of Lassa virus and Mopeia virus were generated by using viruses inactivated by gamma irradiation. This study describes the use of these MAbs to map the antigenic epitopes of each virion protein and to explore the antigenic variability and relationship between arenaviruses from various regions of Africa and South America.
Arenaviruses have been grouped on the basis of morphology, geography, broad serological cross-reactions and to some extent on biologically specific crossneutralization patterns (Johnson et al., 1965; Webb et al., 1969; Murphy et al., 1969; Rowe et al., 1970; Casals et al., 1975). Some sequencing and genetic mapping of Old World arenaviruses has been carried out (Clegg et al., 1983; Gonzalez et al., 1984; Auperin et al., 1986). The development of monoclonal antibodies (MAbs) to lymphocytic choriomeningitis (LCM) virus has allowed exploration of antigenic relationships between certain Old World arenaviruses at the polypeptide level (Buchmeier et al., 1980, 1981, 1987; Howard et al., 1985; Weber et al., 1988). However, this has not been sufficient to define the relationships between these members more precisely. Lassa virus has two segments of single-stranded RNA. The small segment, which codes for the structural proteins of Lassa virus, has an ambisense gene structure (Auperin et al., 1986) as in other arenaviruses (Auperin et al., 1984). Thus the gene for the nucleocapsid protein (NP, 38K) is encoded at the 3' end of viral complementary R N A whereas those for the glycoproteins (GP1, 45K and GP2, 38K) are encoded at the 5' end of the viral R N A (Kiley et al., 1980). The glycoproteins are synthesized as a single precursor (GPC) and cleaved following translation (Clegg & Lloyd, 1983). In this work, carried out without the confines of a maximum containment laboratory, MAbs to the struc0000-9683 ~) 1991 SGM
Methods Virus antigen production. The Josiah strain of Lassa virus isolated from a human patient in Sierra Leone and Mopeia virus isolated from a rodent trapped in Mozambique, were used as antigens for MAb production. Viruses were grown and purified as previously described (Gonzalez et al., 1984). Extracellular virus was concentrated, precipitated and subjected to equilibrium centrifugation on continuous 30% glycerol/45 % potassium tartrate gradients. The virus band was further purified by 20% to 7 0 ~ sucrose gradient centrifugation. All work on infectious virus preparation was performed within the maximum containment laboratory at the Centers for Disease Control. Purified virus was inactivated by gamma irradiation on ice with 2.4 to 2.8 Mrad (Elliott et al., 1982) in a cobalt 60 gamma cell (Model 220, Atomic Energy of Canada). The protein concentration of the inactivated virus was measured and standardized to 150 p.g/0.1 ml. Hybridoma production. Hybridomas were constructed using standard techniques by fusion of SP2/O-Ag 14 mouse myeloma cells with spleen cells from BALB/c mice previously immunized with the inactivated Lassa virus or Mopeia virus antigens as described (K6hler & Milstein, 1976). Cell culture fluid from each well was screened for antibody to Lassa virus and Mopeia virus antigens by indirect immunofluorescence assay (IFA) (Wulff et al., 1975). Cells from antibody-containing wells were cloned by limiting dilution in 96-well culture plates. Ascitic fluids were produced in BALB/c mice by intraperitoneal inoculation.
550
S. L. Ruo and others
The IgG subclasses of MAbs were determined by modified ELISA procedures using hybridoma cell culture fluids and a 96-well plate coated with purified IgG subclass-specific immunoglobulins (Miles Laboratories).
Preparation and testing of antigens by immunofluorescence. Sixty-four isolates of arenaviruses from Africa were grown in Vero E6 ceils. Antigen slides of each virus isolate were prepared as previously described (Johnson et al., 1981) and were inactivated by gamma irradiation. These included 17 human isolates and 10 rodent isolates from West Africa including Sierra Leone, 10 human isolates from Nigeria three human isolates from Guinea, 10 human isolates from Liberia, two rodent isolates from Mozambique (Wulff et al., 1975), three rodent isolates from Zimbabwe and nine rodent isolates from the Central African Republic. Eight New World arenaviruses (Flexal, Brazil; Parana, Paraguay; Junin, Argentina; Tamiami, southern Florida; Pichinde, Colombia; Tacaribe, Trinidad; Amapari, Brazil; Machupo, Bolivia) collected over the past 20 years, as well as LCM virus (Europe, Asia, Africa and North and South America), were similarly prepared for evaluation. IFA asssays were performed by adding drops of MAbs to acetonefixed antigen slide wells, incubating at room temperature in a humidified chamber, washing in phosphate-buffered saline (PBS) and then adding rabbit anti-mouse IgG fluorescein isothiocyanate conjugate (Miles-Yeda). The slides were washed and examined for specific fluorescence (Wulff et al., 1975). Neutralization test. The plaque reduction neutralization titre (NT) of each MAb was measured by mixing serial 10-fold dilutions of antibody with 200 p.f.u, of virus and incubating for 1 h at 37 °C in a water-bath. Vero E6 cells were inoculated with virus-antibody mixtures in six-well plates for 1 h at 37 °C and overlaid with 1 ~ agarose in BME medium. After 4 to 5 days incubation, plate wells were overlaid with 1 ~ Noble agar in Hanks' balanced salt solution with 0.01 ~ neutral red. Plaques were counted and a 50% plaque reduction compared with controls indicated positive neutralization.
buffer at 1 ixg per well and incubated overnight at 4 °C in a 96-well plate. Fifty ixl of a fourfold dilution of unlabelled competing antibody was mixed with 50 Ixl of a 1 : 100 dilution of HRP-conjugated labelled antibody and allowed to equilibrate in microtitre plates for 2 h at 37 °C. The plates were washed, 125 Ixl per well of substrate was added, the reaction was stopped with 25 gl of 3 M-HC1 after colour developed and absorbance values were measured: Competition was determined by comparing test antibodies to those without competing antibodies. More than 8 0 ~ binding inhibition represented positive competition, less than 3 0 ~ binding inhibition represented negative competition and values between 3 0 ~ and 8 0 ~ represented an intermediate competition.
Results Characteristics o f M A b s
Thirty-three MAbs raised against Lassa and Mopeia viruses were produced. The majority of these were isotypes of IgG1, the remainder were IgG2a (Table 1). The IFA patterns of MAbs on infected E6 cells were basically divided into two groups: one group produced a granular or speckled pattern (Fig. 1 a), whereas the other showed a smooth diffuse staining pattern (Fig. lb). The measurement of the protein specificity of MAbs to homologous viruses by RIP (Fig. 2) and immunoblot-
MAb specificity determination. MAb specificity to viral proteins was determined using radioimmunoprecipitation (RIP) followed by SDSpolyacrylamide gel electrophoresis (Laemmli, 1970). Virus-infected E6 cells labelled with [35S]methionine, [3H]leucine or [3H]glucosamine were lysed in Tris-HC1 buffer containing 1 ~ NP40, 0.5~ sodium deoxycholate and 0-1 ~ SDS. The cell extract was reacted with MAb and the antigen-antibody complexes were precipitated with staphylococcal Protein A bacterial adsorbant (Miles-Yeda). The pelleted protein complexes were dissociated and separated by SDS-PAGE in slab gels which were then processed by autoradiography. Immunoblotting was also performed to determine MAb specificity. Briefly, proteins from purified virus were separated by SDS-PAGE and transferred to nitrocellulose filters (Burnette, 1981). Viral proteins separated on nitrocellulose were incubated with MAbs for 2 h at room temperature. The bound MAbs were then detected by incubating with goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) and colour bands were developed from the reaction in substrate solution as previously described (Tsang et al., 1983). Conjugation of antibodies and competitive binding assay. MAbs were purified from mouse ascitic fluids by precipitation with 5 0 ~ ammonium sulphate followed by separation on a Protein A-Sepharose column (Ey et al., 1978). The concentration of purified IgG immunoglobulin was measured by a Bio-Rad protein determination kit and standardized to 1 mg/ml. Purified antibodies were coupled to HRP by the periodate oxidation method (Nakane et al., 1974) and stored in PBS containing 5 ~ bovine serum albumin at - 7 0 °C. A competitive binding assay based on ELISA was developed to determine the arrangement of antigenic epitopes on viral proteins. Purified whole virus was used as antigen, diluted in ELISA coating
Fig. 1. IFA reactions of MAbs with Vero E6 cells infected with Lassa virus. (a) The speckled pattern formed by MAbs specific to Lassa virus nucleoprotein. (b) The smooth pattern formed by MAbs specific to Lassa virus glycoproteins.
Antigenic relatedness between arenaviruses Table 1. Characteristics of MAbs to Lassa and Mopeia
1
2
3
4
5
6
7
8
9
10
551
11
viruses
Clones Lassa virus 52-158-3 52-129-18 52-159-15 52-93-4 52-54-6 52-264-7 52-273-8 52-189-13 52-74-7 52-134-23 52-64-5 52-161-6 52-53-14 52-85-15 52-73-6 52-135-17 52-85-6 52-121-22 52-272-7 52-195-2 52-216-7 52-265-4 52-138-23 52-179-11 52-154-18
IgG Specificity NT Fluorescence subclass by by pattern by ELISA RIP/immunoblotting PRN*
-GP1 Speckled Speckled Speckled Speckled Speckled Speckled Speckled Speckled Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth
G1 G1 G1 G2a G2a G1 G1 G1 G1 G1 G1 G1 G1 G1 G2a G1 G2b G2a G1 G1 G1 G2a G1 G1 G1
NP NP NP NP/NP NP/NP NP/NP NP NP GP 1 GP1 GP1/GP1 GP1 GP1 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2
Speckled Speckled Speckled Smooth Smooth Smooth Smooth Smooth
G1 G1 G2a G1 G1 G1 G1 G1
NP NP/NP NP GP1 GP1/GP1 GP2 GP2 GP2
- GP2 "t - 24K + + +
Mopeia virus
53-29-1 53-144-3 53-7-3 53-16-11 53-193-5 53-12-16 53-177-10 53-237-5
-GPC -NP
* A titre at a 1:10 dilution with more than 50~ plaque reduction compared to positive controls was counted as a positive result. t', Not determined.
ting (Fig. 3) resulted in 11 M A b s r e a c t i n g a g a i n s t N P , 15 a g a i n s t G P 2 a n d seven a g a i n s t GP1 (Table 1). A l l clones showing the s p e c k l e d I F A s t a i n i n g p a t t e r n r e a c t e d w i t h N P a n d p r e c i p i t a t e d a 2 4 K N P - r e l a t e d p o l y p e p t i d e as a b r e a k d o w n p r o d u c t f r o m infected E6 V e r o cell lysates (Fig. 2). This a p p e a r a n c e o f the N P cleavage f r a g m e n t in R I P assays has b e e n d e s c r i b e d in o t h e r r e p o r t s w h i c h f o u n d t h a t the L a s s a virus N P in the i n f e c t e d cells was host c e l l - d e p e n d e n t (Clegg et al., 1983; H u f e r t et al., 1989). T h o s e M A b s s h o w i n g t h e diffuse I F A s t a i n i n g p a t t e r n r e a c t e d w i t h e i t h e r GP1 or G P 2 a n d also r e a c t e d w i t h G P C (Fig. 2), the i n t r a c e l l u l a r p r e c u r s o r o f g l y c o p r o t e i n p r e s e n t in E6-infected cells as d e s c r i b e d for a r e n a v i r u s e s (Clegg et al., 1983; C o m p a n s et al., 1985). T h u s the p r o t e i n specificity staining p a t t e r n s in I F A for b o t h N P a n d G P clones correlate with the results f r o m
Fig. 2. The specificity of MAbs was determined using RIP of viral proteins from Lassa virus-infected E6 cell extracts labelled with [35S]methionine (lanes 2 to 4), [3H]glucosamine (lanes 5 to 7) or [3H]leucine (lanes 8 to 11) and analysed by SDS-PAGE. Lane 2 shows the proteins of Lassa viruses pelleted by 20~ sucrose cushion centrifugation. Lane 7 shows the RIP of uninfected E6 cell lysates with GP2-specific clone 52-73-6 as a negative control. The NP-specific clones (52-93-4; lane 3) and (52-129-18; lane 4) precipitated the 24K proteins which are NP-related degradation or cleavage fragments as described for other arenaviruses (Clegg et al., 1983; Young et al., 1987; Hufert et al., 1989). Lane 1 contains Mr markers. Lanes 5, 6, 10 and 11 contain GP2-specific clones 52-138-23, 52-73-6, 52-121-22 and 52-85-6, respectively. Lanes 8 and 9 contain GPl-specific clones 52-74-7 and 52134-23, respectively. both RIP and immunoblotting. Only glycoproteinspecific M A b s h a v e s h o w n the a b i l i t y to neutralize at low titres ( T a b l e 1).
Epitope sites defined by M A b s E p i t o p e m a p p i n g was p e r f o r m e d b y c o m p e t i t i v e b i n d i n g assays (Fig. 4). H R P - c o n j u g a t e d M A b s were p r e p a r e d b y the p e r i o d a t e o x i d a t i o n t e c h n i q u e w h i c h allowed covalent coupling o f a n a v e r a g e o f one e n z y m e molecule p e r i m m u n o g l o b u l i n v i a s e c o n d a r y a m i n o groups ( N a k a n e et al., 1974). T h e results o f I F A a n d E L I S A h a v e s h o w n t h a t the b i n d i n g a b i l i t y o f c o n j u g a t e d M A b s was compatible with that of unconjugated MAb competitors. T h u s the a n t i g e n i c sites were assigned by M A b s w i t h similar specificity a n d avidity. T h e results o f these assays clearly r e v e a l e d the p r e s e n c e o f at least four e p i t o p i c sites on N P ( N - a , -b, -c, -d), two sites on G P I ( G l - a , -b) a n d six sites on G P 2 (G2-a, -b, -c, -d, -e, -f) o f L a s s a virus (Table 2).
Cross-reactivities between arenaviruses A p a n e l o f 18 diversely r e a c t i n g M A b s was chosen on the basis o f their i m m u n o f l u o r e s c e n t p a t t e r n w i t h h o m o l o -
552
S. L. Ruo and others
l
2
3
4
5
6
7
l
100
I
I
I
HRP-(52-93-4) N P
52-93-4 52-264-7
80 60 200K--
40
52-273-8 52-159-15 52-129-18 52-189-13 52-158-3
20 0
92-5K--
1
100 -HRP-(52-74-7) GP1
68K - NP
GPI
I
I
I
52-74-7 52-64-5 52-134-23
~, 80 .~ 60
52-57-4
43K-40 0
GP2
~
20 0
52-161-6 52-53-14
-
I 25.7K-Fig. 3. The specificity of MAbs was determined by immunoblotting using purified Lassa virus particles as viral protein antigens, separating by SDS-PAGE, transferring to nitrocellulose filters and reacting with MAbs as described in Methods. Polyclonal immune serum from a monkey infected with Lassa virus (lane 1) was used as a positive control to indicate the location of viral proteins migrating on the slab gels. Normal serum was used as a negative control (lane 2). Lanes 3 and 4, GPl-specific clones 52-74-7 and 52-134-23, respectively; lanes 5 to 7, GP2-specific clones 52-85-15, 52-85-6 and 52-138-23, respectively.
I
I 52-73-6
80
52-135-17
60 40 20 0
52-138-23 52-i79-11 52-154-18 52-272-7
" i
0-02
gous viruses and their reactivity with viruses from different areas of Africa (Table 3). Virus isolates from the West African countries of Sierra Leone, Guinea and Liberia reacted equally well with all of the MAbs raised against the Josiah strain of Lassa virus from Sierra Leone. These viruses were distinguishable from those isolated in Nigeria by one Lassa virus NP-directed MAb (52-158-3) and two Lassa virus GPl-directed MAbs (5274-7 and 52-134-23). Only MAbs raised to GP2s and NPs of Lassa virus cross-reacted with southern African virus isolates from Mozambique and Zimbabwe. On the other hand, the MAbs to Mopeia strain virus showed broad cross-reactivities with virus isolates from all African countries tested. Only one N P clone (53-29-1) was highly specific for southern African virus isolates. The GP1 clones, 53-16-11 and 53-193-5, were capable of distinguishing virus isolates from Nigeria and Central Africa respectively (Table 3). Lassa virus MAbs, 52-86-6 and 52-93-4, which reacted with Central African virus isolates also reacted with all of the southern African virus isolates. In contrast, Mopeia virus MAbs, 53-12-16 and 53-73-6, reacted with
I
100 -
i
I
0.08 0.31 1.25 Immunoglobulin (Ixg)
I
5
Fig. 4. Examples of patterns from competitive binding assays with HRP-conjugated Lassa virus MAbs using the modified ELISA procedure. Serial fourfold dilutions of unconjugated MAbs were mixed with individual HRP-conjugated MAbs at a constant dilution and the degree of binding inhibition was calculated and expressed as percentage competition as described in Methods.The immunoglobulin concentration represents the total amount of unconjugated antibodies tested.
all isolates of Mobala virus from Central Africa but with few virus isolates from West Africa (Table 3). Some MAbs were found to be cross-reactive with representative isolates from all of the known South American arenaviruses (Table 4). GP2 MAbs of both Lassa and Mopeia viruses cross-reacted most broadly with the South American arenaviruses. One NP-specific MAb (53-7-3) was found to cross-react with only two viruses (Pichinde and Tacaribe) (Table 4). Thus the broadest cross-reacting and the most conserved antigens appeared to be located on the GP2 viral proteins in both African and South American arenaviruses. GP1 was the least conserved protein, and NP was somewhat cross-reactive within African arenaviruses.
Antigenic relatedness between arenaviruses
Table
2.
553
Epitope mapping of Lassa virus proteins by competitive binding assay using MAbs Competition reaction with HRP-conjugated M A b *
MAb competitor
NP (1) 52-158-3 (2) 52-129-18 52-159-15 (3) 52-93-4 52-54-6 52-264-7 52-273-8 52-189-13
Epitope site
N-a N-b
1
2
3
-['-t
--
--
-t
+
-
--
-[-
--
4
5
6
7
+ + +
+ + +
+ + +
+ + +
+-t
+
+
+
8
9
10
11
12
13
14
---
-_
N-c __
--
-[-
__
--
-[-
N-d
GPI
(4) 52-74-7 (5) 52-134-23 (6) 52-64-5 (7) 52-57-4 52-161-6 52-53-14 GP2 (8) 52-85-15 52-73-6 52-135-17 (9) 52-85-6 52-121-22 (10) 52-272-7 52-195-2 (11) 52-216-7 (12) 52-265-4 (13) 52-138-23 52-179-11 (14) 52-154-18
G 1-a
Gl-b
G2-a
G2-b G2-c
G2-d
-[-
Jr-
.
+
+
.
.
.
-[-
-[-
.
+
+
+
.
--
.[-
-[-
.
.
.
.
--
-["
"Jr-
.
.
.
.
-_
_
-_
-t+
. .
.
.
.
.
. .
. .
. .
-b +
.
G2-e G2-f
.
.
.
.
.
.
-1-
* The number o f each HRP-conjugated M A b corresponds to the number in parentheses next to the appropriate competitor MAb.
t The binding avidities of all M A b s were tested and standardized to 1 mg/ml. The competition reactions are presented as: + , 80 to 1 0 0 ~ binding inhibition; + , 30 to 8 0 ~ binding inhibition and - , less than 3 0 ~ binding inhibition with 5 pg of the M A b solution. :~., Not tested.
Discussion We have produced a large number of diverse MAbs against two biosafety-level 4 viruses, the Lassa and Mopeia strains of arenaviruses, under non-level 4 conditions by using gamma-irradiated viruses. This method clearly presented the immune system with nonreplicating, antigenically intact viruses with functional epitopes on their surfaces since MAbs to membrane glycoproteins could be obtained. Furthermore, we found that the pattern of immunofluorescence on properly harvested infected cells was a simple and consistent indication of the protein specificity of the antibody. We are currently using these MAbs widely for diagnostic, epidemiological and pathogenetic studies. Although MAbs specific to certain antigenic sites on GP1 and GP2 of Lassa virus had shown neutralizing ability at low titres, we have not found any of the high titre neutralizing MAbs described for other arenaviruses (Parekh & Buchmeier, 1986; Sanchez et al., 1989).
Although immunoprecipitation of infected cell lysates with NP-specific MAbs identified NP-related proteins with an Mr of 24K, we have not observed any nuclear antigen inclusion in IFA assays as reported earlier for Pichinde virus (Young et al., 1987). As we did not perform daily observations in the course of Lassa virus infection, these nuclear antigens might have been missed even if they did occur during infection. However, whether these fragment molecules play a regulatory role in Lassa virus replication as is proposed for other arenaviruses is not clear. We have not found any NPspecific MAbs that substantially cross-reacted with all arenaviruses as another report described (Hufert et al., 1989) Some MAbs raised against two different African arenaviruses react with viruses of broadly separated geographical origins and others only with strains from focal areas. However, viruses collected from Sierra Leone over a period of 10 years from human and rodent species reacted uniformly with a panel of MAbs raised
554
S. L. Ruo and others
T a b l e 3. MAb reactions to the virus isolates of African arenaviruses Human isolates ' Sierra Leone
Guinea
Rodent isolates
Liberia
Sierra Leone
Nigeria
Mozambique
Zimbabwe
Central African Republic
Number of positive reactionst/number of isolates tested
M A b clones* Lassa virus NP 52-158-3 52-273-8 52-54-6 52-93-4
17/17 17/17 17/17 17/17
3/3 3/3 3/3 3/3
10/10 10/t0 10/10 10/10
0/10 10/10 10/10 10/10
10/10 t0/10 10/10 10/10
0/2 1/2 2/2 2/2
0/3 3/3 3/3 3/3
0/9 0/9 9/9 9/9
Lassa virus GP1 52-74-7 52-134-23
16/17 16/17
3/3 3/3
5/10 5/10
0/10 0/10
10/10 10/10
0/2 0/2
0/3 0/3
0/9 0/9
Lassa virus GP2 52-85-15 52-135-17 52-85-6 52-121-22
17/17 17/17 17/17 17/17
3/3 3/3 3/3 3/3
10/10 10/10 10/10 10/10
10/10 10/10 10/10 10/10
10/10 10/10 10/10 10/10
0/2 0/2 2/2 2/2
0/3 0/3 3/3 3/3
0/9 0/9 9/9 9/9
Mopeia virus NP 53-29-1 53-144-3 53-7-3
0/17 17/17 9/17
0/3 3/3 3/3
0/10 10/10 10/10
0/10 10/10 3/10
0/10 10/10 10/10
2/2 2/2 2/2
3/3 3/3 3/3
0/9 8/9 9/9
Mopeia virus GPI 53-16-11 8/17 53-193-5 16/17
3/3 3/3
9/10 10/10
0/10 2/10
7/10 9/10
2/2 2/2
3/3 1/3
1/9 0/9
Mopeia virus GP2 53-12-16 11/17 53-177-10 12/17 53-237-5 11/!7
1/3 3/3 3/3
2/10 9/10 9/10
1/10 4/10 4/10
10/10 10/10 10/10
2/2 2/2 2/2
3/3 2/3 3/3
8/9 8/9 7/9
* MAbs were raised specifically against the Lassa and Mopeia viruses. NP, GP1 and GP2 indicate the specificities of MAbs to viral proteins. t Positive reactions were determined by IFA with antibodies at a 1 : 100 dilution.
T a b l e 4. Cross-reactivity of MAbs with New World arenaviruses Representative MAb* Virus t
52-85-6 L-GP2
Flexal Parana Junin Tamiami Pichinde Tacaribe Amapari Machupo LCMII Lassall Mopeiall
- ~ . 1000 100 100 2000 4000 1000 2000 16000 1000
52-121-22 L-GP2
53-12-16 M-GP2
.
. 2000 1000 4000 1000 2000 500 16000 4000
1000§ . 4000 1000 2000 500 2000 32000
53-237-5 M-GP2
53-7-3 M-NP
-
-
. 100 500 8000
1000 100 8000
* Representative MAbs were selected by their ability to cross-react with the African arenaviruses shown in Table 3. L, Lassa; M, Mopeia. t Plaque-purified viruses were obtained from the CDC collection. ~. A negative value represents a titre less than a 1 : 100 dilution. § A reciprocal titre indicates a positive reaction by IFA. I]Controls used for titre comparisons.
against a Sierra Leone Lassa virus strain. This suggested considerable antigenic stability of this virus locally. Our data showed that the most variable antigenic sites included at least one each on the GP1 and NP proteins. Thus, an MAb (52-74-7) to the GP1 and (52-158-3) to the NP of the Josiah strain of Lassa virus from Sierra Leone identified only local isolates from Sierra Leone or from the immediately adjacent countries. Some epitope sites on both NP and GP2 proteins were conserved among the known African arenaviruses. The GP2 proteins revealed two patterns of conservation. One reacted only with West African strain viruses while another reacted with all representative arenaviruses including South American ones. These observations were clearly in agreement with the findings that sequences of the GPC precursors of arenaviruses have shown extensive conservation of deduced amino acid sequences in GP2 proteins (Buchmeier et al., 1987). Furthermore, epitope sites in this conserved region could be specified by only one amino acid substitution as currently reported (Weber et al.,
Antigenic relatedness between arenaviruses
1988). Further study of these antigenic relationships at the primary amino acid sequence level may be able to delineate in detail their relationships to arenavirus e p i d e m i o l o g y a n d diseases. The cross-reactions demonstrated here show that the C e n t r a l A f r i c a n v i r u s e s are a n t i g e n i c a l l y m o r e closely r e l a t e d to t h e v i r u s e s f r o m S o u t h e r n A f r i c a t h a n to t h e W e s t A f r i c a n a r e n a v i r u s e s . T h i s is in a g r e e m e n t w i t h o l i g o n u c l e o t i d e a n a l y s e s w h i c h suggest t h a t C e n t r a l A f r i c a n v i r u s e s ( M o b a l a strains) are s o m e w h a t c l o s e r g e n e t i c a l l y to S o u t h e r n A f r i c a n a r e n a v i r u s e s ( M o p e i a s t r a i n s ) t h a n to t h e o n e s f r o m W e s t A f r i c a ( G o n z a l e z et al., 1984). O t h e r b i o l o g i c a l d a t a , s u c h as n e u t r a l i z a t i o n tests, are n o t yet a v a i l a b l e o r sufficiently r e l i a b l e to assess these relationships further. Although our data have significantly defined these r e l a t i o n s h i p s at t h e n a t i v e p r o t e i n e p i t o p i c level, w e are n o t a b l e as yet to e l u c i d a t e a p a t t e r n o f a r e n a v i r u s e v o l u t i o n . A n y a p p r o a c h to this p r o b l e m m u s t i n c l u d e studies o f a n t i g e n i c a n d g e n e t i c r e l a t i o n s h i p s b e t w e e n v i r u s e s as w e l l as p e r t i n e n t i n f o r m a t i o n o n t h e e v o l u t i o n of the rodent reservoirs.
References AUPERIN, D. D., ROMANOWSKI,V., GALINSKI,M. & BISHOP,D. H. L. (1984). Sequencing studies of Pichinde arenavirus S RNA indicate a novel coding strategy, an ambisense viral S RNA. Journalof Virology 52, 897-904. AUPERIN, D. D., SASSO,D. R. & MCCORMICK,J. B. (1986). Nucleotide sequence of the glycoprotein gene and intergenic region of the Lassa virus S genome RNA. Virology 154, 155-167. BUCHMEIER, M. J. & PAREYdt, B. S. (1987). Protein structure and expression among arenaviruses. Current Topics in Microbiology and Immunology 133, 41-57. BUCHMEIER, M. J., LEWlCKI, H. A., TOMORI,O. & JOHNSON,K. M. (1980). Monoclonal antibodies to lymphocytic choriomeningitis virus react with pathogenic arenaviruses. Nature, London 288, 48764877. BUCHMEIER, M. J., LEWICKI, H. A., OYEWALE, T. & OLDSTONE, M. B. A. (1981). Monoclonal antibodies to lymphocytic choriomeningitis and Pichinde viruses: generation, characterization, and crossreactivity with other arenaviruses. Virology 113, 73-85. BURNETTE,W. N. (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112, 195-203. CASALS,J., BUCKLEY,S. M. & CEDENO,R. (1975). Antigenic properties of the arenaviruses. Bulletin of the World Health Organization 52, 421-427. CLEGG, J. C. S. & LLOYD, G. (1983). Structural and cell-associated proteins of Lassa virus. Journal of General Virology 64, 1127-1136. COMPANS, R. W. & BISHOP, D. H. L. (1985). Biochemistry of arenaviruses. Current Topics in Microbiology& Immunology 114, 153175. ELLIOTT, L. H., McCORMICK, J. B. & JOHNSON, K. M. (1982). Inactivation of Lassa, Marburg and Ebola viruses by gamma irradiation. Journal of Clinical Microbiology 16, 704--708.
555
EY, P. L., PROWSE,S. J. & JENKIN,C. R. (1978). Isolation of pure IgG1, IgG2a, and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15, 429-436. GONZALEZ, J. P., BUCHMEIER,M. J., MCCORMICK, J. B., ELLIOTT, L. H., MITCHELL, S. W. & KILEY, M. P. (1984). Comparative analysis of several Lassa-like arenavirus isolates from Africa. In Molecular Biology of Negative Strand Viruses, pp. 201-208. Edited by D. H. L. Bishop & R. W. Compans. New York: Elsevier. HOWARD,C. R., LEWlCKI,H., ALLISON,L., SALTER,M. & BUCHMEIER, M. J. (1985). Properties and characterization of monoclonal antibodies to Tacaribe virus. Journal of General Virology 66, 1383 1395. HUFERT, F. T., LUDKE,W. & SCHMITZ,H. (1989). Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Archives of Virology 106, 201-212. JOHNSON, K. M., ELLIOTT, L. H. & HEYMANN, D. L. (1981). Preparation of polyvalent viral immunofluorescent intracellular antigens and use in human sera surveys. Journal of Clinical Microbiology 5, 527-529. JOHNSON,K. M., WIEBENGA,N. H., MACKENZIE,R. B., KUNS, M. L., TAURASO,N. M., SHELOKOV,A., WEBB, P. A., JUSTINES, G. & HERE, H. K. (1965). Virus isolation from human cases of hemorrhagic fever in Bolivia. Proceedings of the Society for Experimental Biology and Medicine 118, 113-118. KILEY, M. P., TOMORI,O., REGNERY,R. L. & JOHNSON,K. M. (1980). Characterization of the arenaviruses Lassa and Mozambique. In Replication of Negative Strand Viruses, pp. 1-9. Edited by D. H. L. Bishop & R. W. Compans. New York: Elsevier. K6HLER, G. & MILSTEN, C. (1976). Derivation of specific antibodyproducing tissue and tumour lines by cell fusion. European Journal of Immunology 6, 511-519. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680685. MURPHY, F. A., WEBB, P. A., JOHNSON, K. M. & WHITFIELD, S. G. (1969). Morphological comparison of Machupo with lymphocytic choriomeningitis virus: basis for a new taxonomic group. Journal of Virology 4, 535-541. NAKANE,P. K. & KAWAOI,A. (1974). Peroxidase-labeled antibody: a new method of conjugation. Journal of Histochemistry and Cytochemistry 22, 1084-1091. PAREKH, B. S. & BUCItMEIER,M. J. (1986). Proteins of lymphocytic choriomeningitis virus: antigenic topography of the viral glycoproteins Virology 153, 168 178. ROWE, W. P., PUGH, W. E., WEBB, P. A. & PETERS, C. J. (1970). Serological relationship of the Tacaribe complex of viruses to lymphocytic choriomeningitis virus. Journal of Virology 5, 289-292. SANCHEZ, A., PIrAT, D. Y., KENYON, R. H., PETERS, C. J., McCORMICK, J. B. & KILEY, M. P. (1989). Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses. Journal of General Virology 70, 1125 1132. TSANG, V. C. W., PERALTA,J. M. & SIMONS,A. R. (1983). Enzymelinked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods in Enzymology 92, 377-391. WEBB, P. A., JOHNSON, K. M. & MACKENZIE, R. B. (1969). The measurement of specific antibodies in Bolivian hemorrhagic fever by neutralization of virus plaques. Proceedings of the Society for Experimental Biology and Medicine 130, 1013-1019. WEBER, E. L. & BUCnMEIER,M. J. (1988). Fine mapping of a peptide sequence containing an antigenic site conserved among arenaviruses. Virology 164, 30-38. WULFF, H. & LANGE,J. V. (1975). Indirect immunofluorescence for the diagnosis of Lassa fever infection. Bulletin of the World Health Organization 52, 429-436. YOUNG, P. R., CHANAS,A. C., LEE, S. R., GOULD,E. A. & HOWARD, C. R. (1987). Localization of an arenavirus protein in the nuclei of infected cells. Journal of General Virology 68, 2465-2470.
(Received 18 May 1990; Accepted 22 November 1990)
Printed in Great Britain
549
Antigenic relatedness between arenaviruses defined at the epitope level by monoclonal antibodies S. L. Ruo,* S. W. Mitchell, M. P. Kiley, L. F. Roumillat, S. P. Fisher-Hoch and J. B. McCormick Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, Georgia 30333, U.S.A.
Monoclonal antibodies (MAbs) were produced against two African arenaviruses, Lassa virus and Mopeia virus. Competitive binding analysis of MAbs identified four antigenic sites on the nucleoprotein (NP), two on glycoprotein 1 (GP1) and six on glycoprotein 2 (GP2) of the Josiah strain of Lassa virus. 64 virus isolates from western, central and southern Africa were all consistently distinguishable by MAbs to certain epitopic sites on GP1, GP2 and NP viral proteins.
Furthermore, MAbs to Lassa virus GP1 and NP uniformly distinguished viruses from the West African countries of Sierra Leone, Liberia and Guinea from those of Nigeria. GP2-directed MAbs to two African arenaviruses reacted broadly with South American arenaviruses demonstrating that an epitopic site on GP2 may be the most highly conserved antigen in the arenavirus group.
Introduction
tural proteins of Lassa virus and Mopeia virus were generated by using viruses inactivated by gamma irradiation. This study describes the use of these MAbs to map the antigenic epitopes of each virion protein and to explore the antigenic variability and relationship between arenaviruses from various regions of Africa and South America.
Arenaviruses have been grouped on the basis of morphology, geography, broad serological cross-reactions and to some extent on biologically specific crossneutralization patterns (Johnson et al., 1965; Webb et al., 1969; Murphy et al., 1969; Rowe et al., 1970; Casals et al., 1975). Some sequencing and genetic mapping of Old World arenaviruses has been carried out (Clegg et al., 1983; Gonzalez et al., 1984; Auperin et al., 1986). The development of monoclonal antibodies (MAbs) to lymphocytic choriomeningitis (LCM) virus has allowed exploration of antigenic relationships between certain Old World arenaviruses at the polypeptide level (Buchmeier et al., 1980, 1981, 1987; Howard et al., 1985; Weber et al., 1988). However, this has not been sufficient to define the relationships between these members more precisely. Lassa virus has two segments of single-stranded RNA. The small segment, which codes for the structural proteins of Lassa virus, has an ambisense gene structure (Auperin et al., 1986) as in other arenaviruses (Auperin et al., 1984). Thus the gene for the nucleocapsid protein (NP, 38K) is encoded at the 3' end of viral complementary R N A whereas those for the glycoproteins (GP1, 45K and GP2, 38K) are encoded at the 5' end of the viral R N A (Kiley et al., 1980). The glycoproteins are synthesized as a single precursor (GPC) and cleaved following translation (Clegg & Lloyd, 1983). In this work, carried out without the confines of a maximum containment laboratory, MAbs to the struc0000-9683 ~) 1991 SGM
Methods Virus antigen production. The Josiah strain of Lassa virus isolated from a human patient in Sierra Leone and Mopeia virus isolated from a rodent trapped in Mozambique, were used as antigens for MAb production. Viruses were grown and purified as previously described (Gonzalez et al., 1984). Extracellular virus was concentrated, precipitated and subjected to equilibrium centrifugation on continuous 30% glycerol/45 % potassium tartrate gradients. The virus band was further purified by 20% to 7 0 ~ sucrose gradient centrifugation. All work on infectious virus preparation was performed within the maximum containment laboratory at the Centers for Disease Control. Purified virus was inactivated by gamma irradiation on ice with 2.4 to 2.8 Mrad (Elliott et al., 1982) in a cobalt 60 gamma cell (Model 220, Atomic Energy of Canada). The protein concentration of the inactivated virus was measured and standardized to 150 p.g/0.1 ml. Hybridoma production. Hybridomas were constructed using standard techniques by fusion of SP2/O-Ag 14 mouse myeloma cells with spleen cells from BALB/c mice previously immunized with the inactivated Lassa virus or Mopeia virus antigens as described (K6hler & Milstein, 1976). Cell culture fluid from each well was screened for antibody to Lassa virus and Mopeia virus antigens by indirect immunofluorescence assay (IFA) (Wulff et al., 1975). Cells from antibody-containing wells were cloned by limiting dilution in 96-well culture plates. Ascitic fluids were produced in BALB/c mice by intraperitoneal inoculation.
550
S. L. Ruo and others
The IgG subclasses of MAbs were determined by modified ELISA procedures using hybridoma cell culture fluids and a 96-well plate coated with purified IgG subclass-specific immunoglobulins (Miles Laboratories).
Preparation and testing of antigens by immunofluorescence. Sixty-four isolates of arenaviruses from Africa were grown in Vero E6 ceils. Antigen slides of each virus isolate were prepared as previously described (Johnson et al., 1981) and were inactivated by gamma irradiation. These included 17 human isolates and 10 rodent isolates from West Africa including Sierra Leone, 10 human isolates from Nigeria three human isolates from Guinea, 10 human isolates from Liberia, two rodent isolates from Mozambique (Wulff et al., 1975), three rodent isolates from Zimbabwe and nine rodent isolates from the Central African Republic. Eight New World arenaviruses (Flexal, Brazil; Parana, Paraguay; Junin, Argentina; Tamiami, southern Florida; Pichinde, Colombia; Tacaribe, Trinidad; Amapari, Brazil; Machupo, Bolivia) collected over the past 20 years, as well as LCM virus (Europe, Asia, Africa and North and South America), were similarly prepared for evaluation. IFA asssays were performed by adding drops of MAbs to acetonefixed antigen slide wells, incubating at room temperature in a humidified chamber, washing in phosphate-buffered saline (PBS) and then adding rabbit anti-mouse IgG fluorescein isothiocyanate conjugate (Miles-Yeda). The slides were washed and examined for specific fluorescence (Wulff et al., 1975). Neutralization test. The plaque reduction neutralization titre (NT) of each MAb was measured by mixing serial 10-fold dilutions of antibody with 200 p.f.u, of virus and incubating for 1 h at 37 °C in a water-bath. Vero E6 cells were inoculated with virus-antibody mixtures in six-well plates for 1 h at 37 °C and overlaid with 1 ~ agarose in BME medium. After 4 to 5 days incubation, plate wells were overlaid with 1 ~ Noble agar in Hanks' balanced salt solution with 0.01 ~ neutral red. Plaques were counted and a 50% plaque reduction compared with controls indicated positive neutralization.
buffer at 1 ixg per well and incubated overnight at 4 °C in a 96-well plate. Fifty ixl of a fourfold dilution of unlabelled competing antibody was mixed with 50 Ixl of a 1 : 100 dilution of HRP-conjugated labelled antibody and allowed to equilibrate in microtitre plates for 2 h at 37 °C. The plates were washed, 125 Ixl per well of substrate was added, the reaction was stopped with 25 gl of 3 M-HC1 after colour developed and absorbance values were measured: Competition was determined by comparing test antibodies to those without competing antibodies. More than 8 0 ~ binding inhibition represented positive competition, less than 3 0 ~ binding inhibition represented negative competition and values between 3 0 ~ and 8 0 ~ represented an intermediate competition.
Results Characteristics o f M A b s
Thirty-three MAbs raised against Lassa and Mopeia viruses were produced. The majority of these were isotypes of IgG1, the remainder were IgG2a (Table 1). The IFA patterns of MAbs on infected E6 cells were basically divided into two groups: one group produced a granular or speckled pattern (Fig. 1 a), whereas the other showed a smooth diffuse staining pattern (Fig. lb). The measurement of the protein specificity of MAbs to homologous viruses by RIP (Fig. 2) and immunoblot-
MAb specificity determination. MAb specificity to viral proteins was determined using radioimmunoprecipitation (RIP) followed by SDSpolyacrylamide gel electrophoresis (Laemmli, 1970). Virus-infected E6 cells labelled with [35S]methionine, [3H]leucine or [3H]glucosamine were lysed in Tris-HC1 buffer containing 1 ~ NP40, 0.5~ sodium deoxycholate and 0-1 ~ SDS. The cell extract was reacted with MAb and the antigen-antibody complexes were precipitated with staphylococcal Protein A bacterial adsorbant (Miles-Yeda). The pelleted protein complexes were dissociated and separated by SDS-PAGE in slab gels which were then processed by autoradiography. Immunoblotting was also performed to determine MAb specificity. Briefly, proteins from purified virus were separated by SDS-PAGE and transferred to nitrocellulose filters (Burnette, 1981). Viral proteins separated on nitrocellulose were incubated with MAbs for 2 h at room temperature. The bound MAbs were then detected by incubating with goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) and colour bands were developed from the reaction in substrate solution as previously described (Tsang et al., 1983). Conjugation of antibodies and competitive binding assay. MAbs were purified from mouse ascitic fluids by precipitation with 5 0 ~ ammonium sulphate followed by separation on a Protein A-Sepharose column (Ey et al., 1978). The concentration of purified IgG immunoglobulin was measured by a Bio-Rad protein determination kit and standardized to 1 mg/ml. Purified antibodies were coupled to HRP by the periodate oxidation method (Nakane et al., 1974) and stored in PBS containing 5 ~ bovine serum albumin at - 7 0 °C. A competitive binding assay based on ELISA was developed to determine the arrangement of antigenic epitopes on viral proteins. Purified whole virus was used as antigen, diluted in ELISA coating
Fig. 1. IFA reactions of MAbs with Vero E6 cells infected with Lassa virus. (a) The speckled pattern formed by MAbs specific to Lassa virus nucleoprotein. (b) The smooth pattern formed by MAbs specific to Lassa virus glycoproteins.
Antigenic relatedness between arenaviruses Table 1. Characteristics of MAbs to Lassa and Mopeia
1
2
3
4
5
6
7
8
9
10
551
11
viruses
Clones Lassa virus 52-158-3 52-129-18 52-159-15 52-93-4 52-54-6 52-264-7 52-273-8 52-189-13 52-74-7 52-134-23 52-64-5 52-161-6 52-53-14 52-85-15 52-73-6 52-135-17 52-85-6 52-121-22 52-272-7 52-195-2 52-216-7 52-265-4 52-138-23 52-179-11 52-154-18
IgG Specificity NT Fluorescence subclass by by pattern by ELISA RIP/immunoblotting PRN*
-GP1 Speckled Speckled Speckled Speckled Speckled Speckled Speckled Speckled Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth
G1 G1 G1 G2a G2a G1 G1 G1 G1 G1 G1 G1 G1 G1 G2a G1 G2b G2a G1 G1 G1 G2a G1 G1 G1
NP NP NP NP/NP NP/NP NP/NP NP NP GP 1 GP1 GP1/GP1 GP1 GP1 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2 GP2
Speckled Speckled Speckled Smooth Smooth Smooth Smooth Smooth
G1 G1 G2a G1 G1 G1 G1 G1
NP NP/NP NP GP1 GP1/GP1 GP2 GP2 GP2
- GP2 "t - 24K + + +
Mopeia virus
53-29-1 53-144-3 53-7-3 53-16-11 53-193-5 53-12-16 53-177-10 53-237-5
-GPC -NP
* A titre at a 1:10 dilution with more than 50~ plaque reduction compared to positive controls was counted as a positive result. t', Not determined.
ting (Fig. 3) resulted in 11 M A b s r e a c t i n g a g a i n s t N P , 15 a g a i n s t G P 2 a n d seven a g a i n s t GP1 (Table 1). A l l clones showing the s p e c k l e d I F A s t a i n i n g p a t t e r n r e a c t e d w i t h N P a n d p r e c i p i t a t e d a 2 4 K N P - r e l a t e d p o l y p e p t i d e as a b r e a k d o w n p r o d u c t f r o m infected E6 V e r o cell lysates (Fig. 2). This a p p e a r a n c e o f the N P cleavage f r a g m e n t in R I P assays has b e e n d e s c r i b e d in o t h e r r e p o r t s w h i c h f o u n d t h a t the L a s s a virus N P in the i n f e c t e d cells was host c e l l - d e p e n d e n t (Clegg et al., 1983; H u f e r t et al., 1989). T h o s e M A b s s h o w i n g t h e diffuse I F A s t a i n i n g p a t t e r n r e a c t e d w i t h e i t h e r GP1 or G P 2 a n d also r e a c t e d w i t h G P C (Fig. 2), the i n t r a c e l l u l a r p r e c u r s o r o f g l y c o p r o t e i n p r e s e n t in E6-infected cells as d e s c r i b e d for a r e n a v i r u s e s (Clegg et al., 1983; C o m p a n s et al., 1985). T h u s the p r o t e i n specificity staining p a t t e r n s in I F A for b o t h N P a n d G P clones correlate with the results f r o m
Fig. 2. The specificity of MAbs was determined using RIP of viral proteins from Lassa virus-infected E6 cell extracts labelled with [35S]methionine (lanes 2 to 4), [3H]glucosamine (lanes 5 to 7) or [3H]leucine (lanes 8 to 11) and analysed by SDS-PAGE. Lane 2 shows the proteins of Lassa viruses pelleted by 20~ sucrose cushion centrifugation. Lane 7 shows the RIP of uninfected E6 cell lysates with GP2-specific clone 52-73-6 as a negative control. The NP-specific clones (52-93-4; lane 3) and (52-129-18; lane 4) precipitated the 24K proteins which are NP-related degradation or cleavage fragments as described for other arenaviruses (Clegg et al., 1983; Young et al., 1987; Hufert et al., 1989). Lane 1 contains Mr markers. Lanes 5, 6, 10 and 11 contain GP2-specific clones 52-138-23, 52-73-6, 52-121-22 and 52-85-6, respectively. Lanes 8 and 9 contain GPl-specific clones 52-74-7 and 52134-23, respectively. both RIP and immunoblotting. Only glycoproteinspecific M A b s h a v e s h o w n the a b i l i t y to neutralize at low titres ( T a b l e 1).
Epitope sites defined by M A b s E p i t o p e m a p p i n g was p e r f o r m e d b y c o m p e t i t i v e b i n d i n g assays (Fig. 4). H R P - c o n j u g a t e d M A b s were p r e p a r e d b y the p e r i o d a t e o x i d a t i o n t e c h n i q u e w h i c h allowed covalent coupling o f a n a v e r a g e o f one e n z y m e molecule p e r i m m u n o g l o b u l i n v i a s e c o n d a r y a m i n o groups ( N a k a n e et al., 1974). T h e results o f I F A a n d E L I S A h a v e s h o w n t h a t the b i n d i n g a b i l i t y o f c o n j u g a t e d M A b s was compatible with that of unconjugated MAb competitors. T h u s the a n t i g e n i c sites were assigned by M A b s w i t h similar specificity a n d avidity. T h e results o f these assays clearly r e v e a l e d the p r e s e n c e o f at least four e p i t o p i c sites on N P ( N - a , -b, -c, -d), two sites on G P I ( G l - a , -b) a n d six sites on G P 2 (G2-a, -b, -c, -d, -e, -f) o f L a s s a virus (Table 2).
Cross-reactivities between arenaviruses A p a n e l o f 18 diversely r e a c t i n g M A b s was chosen on the basis o f their i m m u n o f l u o r e s c e n t p a t t e r n w i t h h o m o l o -
552
S. L. Ruo and others
l
2
3
4
5
6
7
l
100
I
I
I
HRP-(52-93-4) N P
52-93-4 52-264-7
80 60 200K--
40
52-273-8 52-159-15 52-129-18 52-189-13 52-158-3
20 0
92-5K--
1
100 -HRP-(52-74-7) GP1
68K - NP
GPI
I
I
I
52-74-7 52-64-5 52-134-23
~, 80 .~ 60
52-57-4
43K-40 0
GP2
~
20 0
52-161-6 52-53-14
-
I 25.7K-Fig. 3. The specificity of MAbs was determined by immunoblotting using purified Lassa virus particles as viral protein antigens, separating by SDS-PAGE, transferring to nitrocellulose filters and reacting with MAbs as described in Methods. Polyclonal immune serum from a monkey infected with Lassa virus (lane 1) was used as a positive control to indicate the location of viral proteins migrating on the slab gels. Normal serum was used as a negative control (lane 2). Lanes 3 and 4, GPl-specific clones 52-74-7 and 52-134-23, respectively; lanes 5 to 7, GP2-specific clones 52-85-15, 52-85-6 and 52-138-23, respectively.
I
I 52-73-6
80
52-135-17
60 40 20 0
52-138-23 52-i79-11 52-154-18 52-272-7
" i
0-02
gous viruses and their reactivity with viruses from different areas of Africa (Table 3). Virus isolates from the West African countries of Sierra Leone, Guinea and Liberia reacted equally well with all of the MAbs raised against the Josiah strain of Lassa virus from Sierra Leone. These viruses were distinguishable from those isolated in Nigeria by one Lassa virus NP-directed MAb (52-158-3) and two Lassa virus GPl-directed MAbs (5274-7 and 52-134-23). Only MAbs raised to GP2s and NPs of Lassa virus cross-reacted with southern African virus isolates from Mozambique and Zimbabwe. On the other hand, the MAbs to Mopeia strain virus showed broad cross-reactivities with virus isolates from all African countries tested. Only one N P clone (53-29-1) was highly specific for southern African virus isolates. The GP1 clones, 53-16-11 and 53-193-5, were capable of distinguishing virus isolates from Nigeria and Central Africa respectively (Table 3). Lassa virus MAbs, 52-86-6 and 52-93-4, which reacted with Central African virus isolates also reacted with all of the southern African virus isolates. In contrast, Mopeia virus MAbs, 53-12-16 and 53-73-6, reacted with
I
100 -
i
I
0.08 0.31 1.25 Immunoglobulin (Ixg)
I
5
Fig. 4. Examples of patterns from competitive binding assays with HRP-conjugated Lassa virus MAbs using the modified ELISA procedure. Serial fourfold dilutions of unconjugated MAbs were mixed with individual HRP-conjugated MAbs at a constant dilution and the degree of binding inhibition was calculated and expressed as percentage competition as described in Methods.The immunoglobulin concentration represents the total amount of unconjugated antibodies tested.
all isolates of Mobala virus from Central Africa but with few virus isolates from West Africa (Table 3). Some MAbs were found to be cross-reactive with representative isolates from all of the known South American arenaviruses (Table 4). GP2 MAbs of both Lassa and Mopeia viruses cross-reacted most broadly with the South American arenaviruses. One NP-specific MAb (53-7-3) was found to cross-react with only two viruses (Pichinde and Tacaribe) (Table 4). Thus the broadest cross-reacting and the most conserved antigens appeared to be located on the GP2 viral proteins in both African and South American arenaviruses. GP1 was the least conserved protein, and NP was somewhat cross-reactive within African arenaviruses.
Antigenic relatedness between arenaviruses
Table
2.
553
Epitope mapping of Lassa virus proteins by competitive binding assay using MAbs Competition reaction with HRP-conjugated M A b *
MAb competitor
NP (1) 52-158-3 (2) 52-129-18 52-159-15 (3) 52-93-4 52-54-6 52-264-7 52-273-8 52-189-13
Epitope site
N-a N-b
1
2
3
-['-t
--
--
-t
+
-
--
-[-
--
4
5
6
7
+ + +
+ + +
+ + +
+ + +
+-t
+
+
+
8
9
10
11
12
13
14
---
-_
N-c __
--
-[-
__
--
-[-
N-d
GPI
(4) 52-74-7 (5) 52-134-23 (6) 52-64-5 (7) 52-57-4 52-161-6 52-53-14 GP2 (8) 52-85-15 52-73-6 52-135-17 (9) 52-85-6 52-121-22 (10) 52-272-7 52-195-2 (11) 52-216-7 (12) 52-265-4 (13) 52-138-23 52-179-11 (14) 52-154-18
G 1-a
Gl-b
G2-a
G2-b G2-c
G2-d
-[-
Jr-
.
+
+
.
.
.
-[-
-[-
.
+
+
+
.
--
.[-
-[-
.
.
.
.
--
-["
"Jr-
.
.
.
.
-_
_
-_
-t+
. .
.
.
.
.
. .
. .
. .
-b +
.
G2-e G2-f
.
.
.
.
.
.
-1-
* The number o f each HRP-conjugated M A b corresponds to the number in parentheses next to the appropriate competitor MAb.
t The binding avidities of all M A b s were tested and standardized to 1 mg/ml. The competition reactions are presented as: + , 80 to 1 0 0 ~ binding inhibition; + , 30 to 8 0 ~ binding inhibition and - , less than 3 0 ~ binding inhibition with 5 pg of the M A b solution. :~., Not tested.
Discussion We have produced a large number of diverse MAbs against two biosafety-level 4 viruses, the Lassa and Mopeia strains of arenaviruses, under non-level 4 conditions by using gamma-irradiated viruses. This method clearly presented the immune system with nonreplicating, antigenically intact viruses with functional epitopes on their surfaces since MAbs to membrane glycoproteins could be obtained. Furthermore, we found that the pattern of immunofluorescence on properly harvested infected cells was a simple and consistent indication of the protein specificity of the antibody. We are currently using these MAbs widely for diagnostic, epidemiological and pathogenetic studies. Although MAbs specific to certain antigenic sites on GP1 and GP2 of Lassa virus had shown neutralizing ability at low titres, we have not found any of the high titre neutralizing MAbs described for other arenaviruses (Parekh & Buchmeier, 1986; Sanchez et al., 1989).
Although immunoprecipitation of infected cell lysates with NP-specific MAbs identified NP-related proteins with an Mr of 24K, we have not observed any nuclear antigen inclusion in IFA assays as reported earlier for Pichinde virus (Young et al., 1987). As we did not perform daily observations in the course of Lassa virus infection, these nuclear antigens might have been missed even if they did occur during infection. However, whether these fragment molecules play a regulatory role in Lassa virus replication as is proposed for other arenaviruses is not clear. We have not found any NPspecific MAbs that substantially cross-reacted with all arenaviruses as another report described (Hufert et al., 1989) Some MAbs raised against two different African arenaviruses react with viruses of broadly separated geographical origins and others only with strains from focal areas. However, viruses collected from Sierra Leone over a period of 10 years from human and rodent species reacted uniformly with a panel of MAbs raised
554
S. L. Ruo and others
T a b l e 3. MAb reactions to the virus isolates of African arenaviruses Human isolates ' Sierra Leone
Guinea
Rodent isolates
Liberia
Sierra Leone
Nigeria
Mozambique
Zimbabwe
Central African Republic
Number of positive reactionst/number of isolates tested
M A b clones* Lassa virus NP 52-158-3 52-273-8 52-54-6 52-93-4
17/17 17/17 17/17 17/17
3/3 3/3 3/3 3/3
10/10 10/t0 10/10 10/10
0/10 10/10 10/10 10/10
10/10 t0/10 10/10 10/10
0/2 1/2 2/2 2/2
0/3 3/3 3/3 3/3
0/9 0/9 9/9 9/9
Lassa virus GP1 52-74-7 52-134-23
16/17 16/17
3/3 3/3
5/10 5/10
0/10 0/10
10/10 10/10
0/2 0/2
0/3 0/3
0/9 0/9
Lassa virus GP2 52-85-15 52-135-17 52-85-6 52-121-22
17/17 17/17 17/17 17/17
3/3 3/3 3/3 3/3
10/10 10/10 10/10 10/10
10/10 10/10 10/10 10/10
10/10 10/10 10/10 10/10
0/2 0/2 2/2 2/2
0/3 0/3 3/3 3/3
0/9 0/9 9/9 9/9
Mopeia virus NP 53-29-1 53-144-3 53-7-3
0/17 17/17 9/17
0/3 3/3 3/3
0/10 10/10 10/10
0/10 10/10 3/10
0/10 10/10 10/10
2/2 2/2 2/2
3/3 3/3 3/3
0/9 8/9 9/9
Mopeia virus GPI 53-16-11 8/17 53-193-5 16/17
3/3 3/3
9/10 10/10
0/10 2/10
7/10 9/10
2/2 2/2
3/3 1/3
1/9 0/9
Mopeia virus GP2 53-12-16 11/17 53-177-10 12/17 53-237-5 11/!7
1/3 3/3 3/3
2/10 9/10 9/10
1/10 4/10 4/10
10/10 10/10 10/10
2/2 2/2 2/2
3/3 2/3 3/3
8/9 8/9 7/9
* MAbs were raised specifically against the Lassa and Mopeia viruses. NP, GP1 and GP2 indicate the specificities of MAbs to viral proteins. t Positive reactions were determined by IFA with antibodies at a 1 : 100 dilution.
T a b l e 4. Cross-reactivity of MAbs with New World arenaviruses Representative MAb* Virus t
52-85-6 L-GP2
Flexal Parana Junin Tamiami Pichinde Tacaribe Amapari Machupo LCMII Lassall Mopeiall
- ~ . 1000 100 100 2000 4000 1000 2000 16000 1000
52-121-22 L-GP2
53-12-16 M-GP2
.
. 2000 1000 4000 1000 2000 500 16000 4000
1000§ . 4000 1000 2000 500 2000 32000
53-237-5 M-GP2
53-7-3 M-NP
-
-
. 100 500 8000
1000 100 8000
* Representative MAbs were selected by their ability to cross-react with the African arenaviruses shown in Table 3. L, Lassa; M, Mopeia. t Plaque-purified viruses were obtained from the CDC collection. ~. A negative value represents a titre less than a 1 : 100 dilution. § A reciprocal titre indicates a positive reaction by IFA. I]Controls used for titre comparisons.
against a Sierra Leone Lassa virus strain. This suggested considerable antigenic stability of this virus locally. Our data showed that the most variable antigenic sites included at least one each on the GP1 and NP proteins. Thus, an MAb (52-74-7) to the GP1 and (52-158-3) to the NP of the Josiah strain of Lassa virus from Sierra Leone identified only local isolates from Sierra Leone or from the immediately adjacent countries. Some epitope sites on both NP and GP2 proteins were conserved among the known African arenaviruses. The GP2 proteins revealed two patterns of conservation. One reacted only with West African strain viruses while another reacted with all representative arenaviruses including South American ones. These observations were clearly in agreement with the findings that sequences of the GPC precursors of arenaviruses have shown extensive conservation of deduced amino acid sequences in GP2 proteins (Buchmeier et al., 1987). Furthermore, epitope sites in this conserved region could be specified by only one amino acid substitution as currently reported (Weber et al.,
Antigenic relatedness between arenaviruses
1988). Further study of these antigenic relationships at the primary amino acid sequence level may be able to delineate in detail their relationships to arenavirus e p i d e m i o l o g y a n d diseases. The cross-reactions demonstrated here show that the C e n t r a l A f r i c a n v i r u s e s are a n t i g e n i c a l l y m o r e closely r e l a t e d to t h e v i r u s e s f r o m S o u t h e r n A f r i c a t h a n to t h e W e s t A f r i c a n a r e n a v i r u s e s . T h i s is in a g r e e m e n t w i t h o l i g o n u c l e o t i d e a n a l y s e s w h i c h suggest t h a t C e n t r a l A f r i c a n v i r u s e s ( M o b a l a strains) are s o m e w h a t c l o s e r g e n e t i c a l l y to S o u t h e r n A f r i c a n a r e n a v i r u s e s ( M o p e i a s t r a i n s ) t h a n to t h e o n e s f r o m W e s t A f r i c a ( G o n z a l e z et al., 1984). O t h e r b i o l o g i c a l d a t a , s u c h as n e u t r a l i z a t i o n tests, are n o t yet a v a i l a b l e o r sufficiently r e l i a b l e to assess these relationships further. Although our data have significantly defined these r e l a t i o n s h i p s at t h e n a t i v e p r o t e i n e p i t o p i c level, w e are n o t a b l e as yet to e l u c i d a t e a p a t t e r n o f a r e n a v i r u s e v o l u t i o n . A n y a p p r o a c h to this p r o b l e m m u s t i n c l u d e studies o f a n t i g e n i c a n d g e n e t i c r e l a t i o n s h i p s b e t w e e n v i r u s e s as w e l l as p e r t i n e n t i n f o r m a t i o n o n t h e e v o l u t i o n of the rodent reservoirs.
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(Received 18 May 1990; Accepted 22 November 1990)
