C. R. HOWARD AND OTHERS. Table 1. Immunofluorescence analysis of monoclonal antibodies to Tacaribe virus. (a) Antibodies reactive against homologous ...
J. gen. Virol. (1985), 66, 1383-1395.
Printed in Great Britain
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Key words: arenaviruses/Junin virus/Tacaribe ~,irus/monoclonal antibodies
Properties and Characterization of Monoclonal Antibodies to Tacaribe Virus By C. R. H O W A R D , 1. H. L E W I C K I , 2 L. A L L I S O N , 1 M. S A L T E R 1 AND M. J. B U C H M E I E R 2
1 Department of Medical Microbiology, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, U.K. and 2 Department of Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037, U.S.A. (Accepted 4 April 1985) SUMMARY
Monoclonal antibodies prepared against Tacaribe and Junin viruses have been used to define further the serological relationships between arenaviruses of the Tacaribe complex. A close relationship was found between these two viruses and the heterologous Amapari and Machupo viruses, with Pichinde virus and Parana virus being more distantly related. Among the antibodies specific for Tacaribe virus, five were found to react with viral antigens at the surface of infected cells and to neutralize virus infectivity in vitro. These five antibodies could be differentiated by competitive immunoassay as recognizing at least two antigenically distinct epitopes. The kinetics of reaction between antibody and virus were examined for all five neutralizing antibodies. One antibody (2.25.4) effectively neutralized all infectious virus. The remaining four directed against a second epitope gave significant persistent fractions which could be reduced by addition of complement, anti-mouse immunoglobulin, or antibody 2.25.4. Variants of Tacaribe virus resistant to neutralization by antibody 2.25.4 were obtained by growth in the presence of this antibody and neutralization kinetics were reexamined using the heterologous monoclonal neutralizing antibodies. Several different neutralization profiles were obtained, suggesting that point mutations resulted in conformational changes at topographically selected distinct epitopes recognized by the remaining antibodies. INTRODUCTION
The Tacaribe complex of the family Arenaviridae presently contains eight viruses, all of which serologically cross-react to varying degrees with Tacaribe virus, the type member of the group (Wulffet al., 1978). Other members include Pichinde, Amapari and Parana viruses, all of which cause persistent infections in their natural rodent hosts but are non-pathogenic for man. Machupo and Junin viruses are also members of the complex, being the causative agents of Bolivian and Argentinian haemorrhagic fevers respectively. Tacaribe virus in common with other arenaviruses contains large (L) and small (S) RNA genome segments (Vezza et al., 1978). The virus contains a major internal nucleocapsid protein (N) and has been reported to contain a single glycoprotein species in its outer envelope (Gard et al., 1977). This is in contrast to other arenaviruses so far characterized including lymphocytic choriomeningitis (LCM) virus, Pichinde and Junin viruses, all of which contain two glycoprotein moieties in the viral envelope (Ramos et al., 1972; Vezza et al., 1978; Grau et al., 1981; Buchmeier et al., 1978). Extensive serological cross-reactivity among members of the Tacaribe complex is readily demonstrable by complement fixation techniques (Casals et al., 1975; Wulff et al., 1978) although the degree of relatedness between individual members has been difficult to assess using convalescent or hyperimmune antisera. However, Junin, Machupo, Amapari and Tacaribe viruses appear to be particularly closely related. All available evidence indicates that the 0000-6574 © 1985 SGM
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c . R. HOWARD AND OTHERS
c o m p l e m e n t - f i x i n g antigen is associated w i t h the internal N p o l y p e p t i d e ( B u c h m e i e r et al., 1977). T h e neutralization test is m u c h m o r e specific, showing no cross-neutralization b e t w e e n viruses of the T a c a r i b e c o m p l e x w h i c h are otherwise closely related by c o m p l e m e n t fixation. F o r e x a m p l e , J u n i n and M a c h u p o viruses are quite distinct ( J o h n s o n et al., 1973), a l t h o u g h there is s o m e e v i d e n c e to suggest that J u n i n virus is weakly neutralized by T a c a r i b e i m m u n e s e r u m ( H e n d e r s o n & D o w n s , 1965; W e i s s e n b a c h e r et al., 1975/1976). F u r t h e r m o r e , n e i t h e r guineapigs nor m a r m o s e t m o n k e y s inoculated w i t h T a c a r i b e virus show signs o f disease but they are p r o t e c t e d against challenge w i t h the n o r m a l l y lethal J u n i n virus, an o b s e r v a t i o n w h i c h has led to the suggestion that T a c a r i b e virus m a y be a possible c a n d i d a t e v a c c i n e against A r g e n t i n e h a e m o r r h a g i c fever ( W e i s s e n b a c h e r et al., 1975/1976, 1982). O n e reason for this p r o t e c t i o n m a y be that T a c a r i b e virus primes an i m m u n e response to J u n i n virus as a result o f the a n t i g e n i c similarity b e t w e e n t h e m (Coto et al., 1980). H o w e v e r , the n a t u r e of this cross-protection and the antigen(s) i n v o l v e d are as yet u n c h a r a c t e r i z e d . T h e d e v e l o p m e n t o f m o n o c l o n a l a n t i b o d i e s specific for these viruses w h i c h was necessary for the analysis o f this a n t i g e n i c relationship is r e p o r t e d in this paper. T h e s e antibodies h a v e also been used to clarify further the extent of a n t i g e n i c cross-reactivity b e t w e e n m e m b e r s o f the T a c a r i b e c o m p l e x , thereby e x t e n d i n g p r e v i o u s results o b t a i n e d w i t h m o n o c l o n a l antibodies against L C M and P i c h i n d e viruses ( B u c h m e i e r et al., 1980, 1981). N e u t r a l i z i n g a n t i b o d y against m a n y arenaviruses can only be d e t e c t e d w i t h difficulty ( C h a n a s et al., 1980) and the finding o f m o n o c l o n a l a n t i b o d i e s w i t h neutralizing properties against T a c a r i b e virus p r o m p t e d a detailed e x a m i n a t i o n o f these reagents and their use to p r e p a r e stable v a r i a n t s resistant to neutralization. METHODS Virus and viral growth. Tacaribe (strain TRVL 11573) and Parana viruses were obtained from the Center for Disease Control, Atlanta, Ga., U.S.A. (courtesy of Drs K. Johnson and J. McCormick) as a suckling mouse brain suspension. Attenuated Junin virus (XJ-C 13 strain) was obtained from Dr G. Eddy, U.S. Army Research Institute of Infectious Diseases, Fort Derrick, Md., U.S.A. When required, extracellular virus was purified from roller bottle cultures of infected BHK-2I cells and purified as described previously (Buchmeier et at., 1978). Immunization andfi~sion. BALB/c mice selected at 4 to 6 weeks of age from the Scripps Clinic and Research Foundation breeding colony were primed by intraperitoneal injection of 103 p.f.u, of virus. Primed animals received a further 103 p.f.u. 1 week later. All animals immunized with Tacaribe virus contained circulating antibody detectable by immunofluorescence after 4 weeks. Mice showing the highest titres were selected and boosted by intraperitoneal injection of 0.3 ml of a 10% (v/v) saline extract of infected cells on three successive days prior to sacrifice. Animals primed initially with two doses of Junin X J-C13 at 1000 p.f.u, per dose failed to produce a detectable antibody response. These animals were rested for 3 months and then further immunized, at first with 10~ p.f.u., and then with 105 p.f.u. 3, 6, 7 and 8 days later. Mice were sacrificed 24 h later. Serum from the Junin virusinoculated mice used for fusion were confirmed retrospectively as having antibody to the virus. Spleens selected for fusion were prepared as described by K6hler & Milstein (1975) and splenocytes fused with the non-secreting P3-X63-Ag8-6531 line of mouse plasmacytoma cells. Detection of hybrid cell colonies secreting antibody was accomplished by indirect immunofluorescence using both acetone-fixed infected L or Vero cell substrates and viable cell suspensions as previously described for the detection of LCM virus antibody (Buchmeier & Oldstone, 1978; Collins et al., 1982). Positive colonies were subsequently subcultured and cloned by limiting dilution as described previously (Buchmeier et al., 1981) and cloned cultures were grown on ascites using BALB/c mice primed with Pristane (Aldrich). Virus assay and neutralization. Both Tacaribe and Junin viruses were quantified by plaque assay using Vero cells. The procedure was essentially as described by Mann et al. (1980). In some early experiments, CM-cellulose was used as a liquid overlay, but plaques were consistently of smaller diameter after 8 days of incubation than they were when agarose was used. Aliquots of supernatant fluid from antibody-producing hybridoma cell cultures were initially screened for neutralization in vitro by measurement of virus titre reduction in the presence of a constant amount of antibody. Mixtures containing 0.1 ml of antibody-containing fluid and 0.1 ml of virus diluted in 10-fold increments were made directly in 24-well tissue culture plates and left in the dark for 30 min at 37 °C. Vero ceils (5 × l0 s) in 1 ml growth medium [Medium 199 containing 5 ~ (v/v) foetal calf serum and antibiotics] were added to each well and plates further incubated at 37 °C for 3 h. The medium was then withdrawn and a CM-cellulose overlay added. The
Tacaribe virus m o n o c l o n a l antibodies
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extent of virus neutralization was recorded as the log~0 reduction in virus titre observed after plaque visualization 7 days later. Mouse ascites containing polyclonal antibodies to Tacaribe virus or hamster immune serum to Junin virus were used as positive control reagents where appropriate (kindly provided by Dr K. Johnson). Neutralizing antibodies against Tacaribe virus were examined further in order to quantify the rate of reaction. Neutralization kinetics were measured by incubating dilutions of heat-inactivated ascites containing antibody with approximately 5 x l0 s p.f.u, of Tacaribe virus in equal volumes. All dilutions were pre-warmed at 30 °C and aliquots of each mixture were withdrawn at various times after mixing. Virus-antibody reactions were stopped by an immediate 1:100 dilution into Medium 199 containing 2% (v/v) foetal calf serum held on ice; residual virus infectivity was quantified on Vero cell monolayers. Selection o f virus variants. Approximately 107 p.f.u, of freshly cloned Tacaribe virus was mixed with a 1:500 final dilution of monoclonal antibody 2.25.4 for 15 min at 37 °C. The mixture was then plated on Vero cells at high dilution under an agar overlay containing the same dilution of antibody. The plates were examined by neutral red staining 7 days later and well-separated plaques representing virus that had escaped neutralization were recloned three times in the presence of antibody and stocks grown in BHK-21 cells for further analysis. Variants were selected that retained reactivity in kinetic neutralization experiments with a mouse hyperimmune antiserum but were resistant to antibody 2.25.4. Immune precipitation. The specificity of monoclonal antibodies against Tacaribe virus was investigated by immune precipitation of [3SS]methionine-labelled viral proteins from cell extracts as described previously (Buchmeier et aL, 1981 ; Collins et al., 1982). The resulting immune complexes were removed by addition of formalin-fixed Staphylococcus aureus and analysed on 10.5% SDS-poiyacrylamide gels (Buchmeier et al., I978). Solid-phase competition radioimmunoassay. Purified Tacaribe virus was bound to the surface of flexible polyvinyl microtitre plates (Cooke Engineering) by inoculation overnight at room temperature in 0.1 M-bicarbonate buffer pH 9.0. Approximately 1 gg of viral protein per well was previously determined as optimal for detection of specific antibody. The wells were then washed and treated with 0.25 % bovine serum albumin (BSA~2 % foetal calf serum in PBS to block remaining sites. Individual monoclonal antibodies were purified by Protein A affinity chromatography (Ey et al., 1978) and radiolabelled with Bolton & Hunter reagent (Amersham) to a specific activity of 0-1 to 0-3 ktCi/~tg. Competition experiments were performed by adding purified monoclonal antibody IgG diluted fivefold in 0.25% BSA PBS directly into coated wells and leaving for 30 rain at room temperature. A fixed amount of radiolabelled antibody was then added to all wells and incubation continued for 3 h at 37 °C. The wells were then washed three times with 0.1% Tween 20-PBS, dried, and counted by gamma spectroscopy.
RESULTS
Production of monoclonal antibodies A total of 21 cell clones secreting antibodies to Tacaribe virus were obtained from a total of 112 hybrid cultures after fusion of immune spleen cells with P3-X63-Ag8-6531 plasmacytoma cells. Positive cultures were identified by immunofluorescence analysis of tissue culture fluids using fixed, infected L-cell substrates. No reactions were recorded using uninfected cell substrates as negative controls. Of the 21 cell cultures, three were dispensed with on grounds of poor antibody production. The remaining 18 were cloned, re-checked for antibody production and hybridoma cells injected into Pristane-treated mice for the production of ascites. Of these, the majority secreted antibody of the IgG2a subclass (12), and four produced antibody of IgG type 1. The remaining two produced IgG2b and IgM antibodies respectively (Table 1). In one case (Tacaribe antibody 2.14.D4) it was necessary to re-clone the culture to establish a satisfactory cell line. Following the immunization protocol outlined in Methods, monoclonal antibodies to the attenuated X J-El 3 Junin virus were produced in seven of a total of 120 cultures. All monoclonal antibodies to Junin were of the IgG type, three of subclass 2a, three of subclass 1 and one of subclass 2b (Table 2).
Properties of monoclonal antibodies The initial screening of cultures by immunofluorescence segregated the antibodies to Tacaribe virus into three groups. The first of these produced both a fine granular cytoplasmic fluorescence and a reaction at the plasma membrane (antibodies 2.2. I to 2.82.2, Table 1 a). All five antibodies in this group produced a positive immunofluorescence reaction at the surface of unfixed infected cells. The second group of antibodies (2.1.2 to 2.43.1, Table 1a) gave positive
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AND OTHERS
Table 1. Immunofluorescence analysis of monoclonal antibodies to Tacaribe virus (a) A n t i b o d i e s r e a c t i v e a g a i n s t h o m o l o g o u s s u b s t r a t e s only
Ref. no.
Ig subclass
Cytoplasmic fluorescence*
Plasma membrane fluorescence*
2.2.1 2.12.5 2.14.D4 2.25.4 2.82.2 2.1.2 2.7.2 2.29.9 2.30.10 2.31.1 2.69.1 2.84.11 2.43.1
G2a GI G2a G2a G2a G2a G1 G2b G2a G2a G2a M GI
+ + + + + + + + + + + + +
+ + + + + -
(b) A n t i b o d i e s r e a c t i v e a g a i n s t heterologous s u b s t r a t e s Cross-reactionst Ref. no.
Ig subclass
Tacaribe
Junin
Machupo
Pichinde
Amapari
Parana
2.16.2 2.48.3 2.52.2 2.74.3 2. 100.3
G2a G2a G2a G2a G1
320 40000 160000 320000 32000
+~ 4680 + 1620 +
-:~ 500 500
3200 -
10 10000 80000 80000 -
10 -
* E x p e r i m e n t s w e r e p e r f o r m e d using e i t h e r acetone-fixed cells ( c y t o p l a s m i c fluorescence) or ( m e m b r a n e fluorescence) infected L-cell cultures ( B u c h m e i e r et al., 1981). "~ E x p r e s s e d as r e c i p r o c a l o f e n d p o i n t titres o b t a i n e d w i t h ascites fluids. :~ + , Positive (not t i t r a t e d ) ; - , n e g a t i v e .
unfixed
Table 2. Cross-reactions by immunofluorescence with monoclonal antibodies to Junin virus Cross-reactions* Ref. no. 3.6 3.46 3.67 3.69 3.70 3.88 3.95
Ig subclass
Junin
Machupo
Pichinde
Tacaribe
Amapari
Parana
G2b G2a G2a GI GI G2a G1
+ + + + + + +
+ + + + + + +
+ -
+ + + + + + +
-
-
* + , P o s i t i v e ; - , n e g a t i v e . All substrates w e r e acetone-fixed infected m o u s e L-cell cultures ( B u c h m e i e r et al., 198l).
reactions only with acetone-fixed substrates infected with Tacaribe virus. The remaining antibodies were characterized by reactions with acetone-fixed substrates infected with heterologous arenaviruses in the absence of surface fluorescence (Table 1 b). All in this group reacted with Junin and four with Amapari viral antigens. Among these five antibodies, two also reacted with Machupo viruses and one additionally recognized Pichinde antigens (antibody 2.48.3). Parana virus-infected cells were only recognized by antibody 2.16.2 which failed to react with either Machupo or Pichinde virus. Representative antibodies from the second and third group reacted with the internal N polypeptide of Tacaribe virus as assessed by analysis of immune complexes obtained by reaction of antibody with radiolabelled cell extracts (Fig. I a, c, d,e).
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Tacar~e v~usmonoclonal antibodies ~)
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(d
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G G
Fig. 1. SDS-PAGE analysis of immunoprecipitates formed by the reaction of infected cell extracts with Tacaribe virus monoclonal antibodies giving cytoplasmic immunofluorescent reactions on acetone-fixed cells [(a, c, d, e) antibodies prefixed 2.2, 2.16, 2.74 and 2.7 respectively in Table 1] or surface fluorescence on unfixed cells and neutralization of virus infectivity [(b,J) antibodies prefixed 2.14 and 2.2 respectively]. Both N and G polypeptides were precipitated by mouse hyperimmune serum (g). No precipitation occurred with normal mouse serum (h). (a) to (d) and (e) to (h) represent two separate experiments.
All of the monoclonal antibodies to Tacaribe virus which gave positive reactions at the surface of unfixed infected cells immunoprecipitated the single envelope glycoprotein (G) of this virus (e.g. Fig. 1 b,J). However, these immunoprecipitates also contained the nucleocapsid (N) protein, a finding in common with previous studies of antibodies to arenavirus glycoproteins (Kiley et al., 1981). Attempts to confirm the specificity of these antibodies by Western blotting techniques were unsuccessful, suggesting they are directed against conformation-dependent epitopes. As a control, a polyclonal immune ascitic fluid positive for neutralizing antibodies to Tacaribe virus precipitated both structural proteins (Fig. lg). A limited number of monoclonals to Junin virus strain XJ-C 13 were also prepared to examine the extent of reciprocal cross-reactions by immunofluorescence. All seven clones showed positive reactions against fixed cell substrates infected with the same Junin virus strain. There was no evidence of antibody binding at the cell surface and all showed cross-reactions against other members of the Tacaribe virus complex (Table 2). All gave positive reactions with Tacaribe virus-infected substrates, confirming the close relationship between these viruses. Additionally, all recognized Machupo virus antigens. In contrast to the monoclonal antibodies to Tacaribe virus, no cross-reactions were seen against Amapari or Parana viruses and only one antibody recognized Pichinde virus antigens. Titration analysis using substrates infected with virulent strains of Junin virus showed that these antibodies gave varying titres of reactivity compared to the X J-C13 strain (C. R. Howard & J. I. Maiztegui, unpublished observations).
1388
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1000 100 10 1 0-1 IgG/well (ng) Fig. 2. Solid-phase competition radioimmunoassays using monoclonal antibodies with neutralization properties against Tacaribe virus. Four purified immunoglobulins were 125I-labelled with Bolton & Hunter reagent and each ligand was examined for binding to purified virus in the presence of increasing amounts of heterologous antibodies. Labelled ligands used in the four experiments were (a) 2.2.1, (b) 2.12.5, (c) 2.14. D4 and (d) 2.82.2. Competing antibodies prepared as purified IgG are represented as follows: I , 2.25.1; O, 2.2.1; A, 2.12.5; I-], 2.14.D4; O, 2.82.2.
Competition radioimmunoassay
All five monoclonal antibodies to the Tacaribe virus glycoprotein were radiolabeUed and used as radioactive probes in a solid-phase radioimmunoassay containing whole virus bound to the solid phase as described in Methods. Each antibody was tested in turn for cross-competition with the remaining four antibodies (Fig. 2). Complete cross-reaction was seen between antibodies 2.2.1, 2.82.2, 2.12.5 and 2.14. D4, these being the same groups of antibodies which produced a high level of non-neutralized virus (Fig. 4). Antibody 2.25.1, from a clone similar in properties to 2.25.4, did not cross-compete with any of these antibodies. Attempts to perform the reciprocal experiment whereby antibody 2.25.1 was used as the radioactive probe proved unsuccessful as this antibody lost its ability to bind to virus after radiolabelling using either the Bolton & Hunter reagent, lactoperoxidase, chloramine-T or Iodogen or a variety of other labelling procedures. Hence, a labelled probe was unavailable. Unlabelled 2.25.1 IgG used in competition experiments was positive by immunofluorescence, confirming that reactivity of this antibody had not been lost on purification. Neutralization o f Tacaribe virus with monoclonal antibodies
Aliquots of supernatant fluid from antibody-producing hybridoma cell cultures were screened for neutralization in vitro by measurement of virus titre reduction in the presence of a constant amount of antibody. All five antibodies in the group previously found to react with the surface of Tacaribe virus-infected cells (see Table 1) reduced viral infectivity (Table 3). In contrast, no significant levels of neutralization were found against the heterologous arenaviruses Pichinde and Amapari (data not shown). Although some neutralization was obtained against Junin virus XJ-C13 (up to 0.3 log10 p.f.u, of total plaque number), this was deemed to be not significantly
1389
Tacaribe virus monoclonal antibodies Table 3. Neutralization of inJectivity by monoclonal antibodies to Tacaribe virus Virus neutralized* f
Specificity Envelope glycoprotein
Nucleocapsid protein
Control serum Mouse anti-Tacaribe Mouse anti-Junin
Antibody no. 2.2.1 2.12.2"1" 2.14. D4 2.25.4 2.82.2 2.16.2 2.48.3 2.52.2 2.74.3 2. 100.1 2.1.5 2.7.2 2.29.9 2.30.10 2.31.1
-
-
A
Tacaribe 2.87 1.95 1.83 2.84 1-84 0.10 0.18 0.33 0.20 0'25 0 0"16 0"14 0' 19 0"19
Junin 0.23 0-16 0.02 0.27 0.34
2.39 0
> 1.0 > 3.0
ND~ 0.17 ND ND ND 0"24 ND ND ND ND
* Reduction in log~0 titre compared to control. ~"Clone similar in properties to 2.12.5 (Table 1). :~NO, Not done.
different from the maximum values obtained against Tacaribe virus using antibodies specific for the nucleocapsid. The one-way cross-neutralization observed using polyclonal mouse antisera as positive control was noteworthy : anti-Junin sera failed to neutralize Tacaribe virus but anti-Tacaribe immune ascites neutralized both Junin and the homologous virus, as previously reported by Henderson & Downs (1965). Antibodies against Tacaribe virus found positive for homologous neutralization were examined further in order to quantify the rate of reaction between antibody and homologous virus. Kinetic neutralization showed that the five antibodies differed according to the level of infectivity remaining after 20 min of incubation. At high dilutions, antibody 2.25.4 effectively neutralized all infectious virus within the limit of sensitivity of the assay system; neutralization curves demonstrated an initial lag period followed by linear neutralization of virus with time (Fig. 3). 'Least squares' linear regression analysis of data obtained in the linear region of each neutralization curve showed highly significant values for correlation coefficients (P =
