A topological and functional model of epitopes on the structural glycoprotein of tick-borne encephalitis virus defined by monoclonal antibodies

VIROLOGY126, 525-537 (1983)

A Topological and Functional Model of Epitopes on the Structural Glycoprotein of Tick-Borne Encephalitis Virus Defined by Monoclonal Antibodies F R A N Z X. H E I N Z , 1 R U D O L F B E R G E R , 2 W O L F G A N G AND C H R I S T I A N K U N Z

TUMA,

Institute of Virology, University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria Received October 20, 1982; accepted December 31, 1982 A topological and functional model of eight distinct epitopes on the structural glycoprotein of tick-borne encephalitis (TBE) virus was established by the use of monoclonal antibodies. The unique specificities and spatial relationships of these antibodies were determined by variant analysis, haemagglutination inhibition (HI), neutralization, passive mouse protection, and antibody blocking assays. Seven out of the eight distinct epitopes were shown to be partially linked and to cluster in two antigenically reactive domains (A, B). Each of these domains is inhomogeneous and contains constituents with different serological specificities and functions. Domain A is defined by three HA-inhibiting antibodies, two of which are flavivirus group-reactive, whereas the third is TBE virus subtype specific. Within this domain only the subtype-specific antibody is involved in virus neutralization, thus explaining the observation that neutralization tests with flaviviruses show higher serological specificities than HI tests and that HI tests can be made type and subtype specific by antibody absorption. Domain B is composed of three TBE-complex reactive epitopes, and the corresponding antibodies inhibit HA and neutralize the virus. A fourth epitope linked to this domain is neither involved in HA nor in neutralization and the same holds true for a subtype-specific epitope which is topologically independent of domains A and B. Each of two different nonneutralizing antibodies was capable of blocking the binding of distinct neutralizing antibodies. All eight epitopes are indistinguishably present on strains of the western subtype of TBE virus isolated all over Europe in different years from different hosts, thus again confirming the great stability of this virus. INTRODUCTION

al., 1980) w h i c h h a s p r o v e n to be w e l l t o l e r a t e d a n d efficacious ( K u n z et al., 1980a, b). S i m i l a r to o t h e r f l a v i v i r u s e s , T B E v i r u s contains three structural proteins termed E, C, a n d M a c c o r d i n g t o a n e w l y p r o p o s e d n o m e n c l a t u r e ( W e s t a w a y , et al., 1980); the corresponding molecular weights are 53,000, 15,000, a n d 7,500 ( H e i n z a n d K u n z , 1981). E r e p r e s e n t s t h e o n l y s t r u c t u r a l g l y coprotein which carries haemagglutinat i o n ( H A ) a c t i v i t y , a n d , t o g e t h e r w i t h M, is l o c a t e d in t h e l i p o p r o t e i n e n v e l o p e . C is t h e o n l y p r o t e i n c o n s t i t u e n t of t h e n u c l e o c a p s i d ( H e i n z a n d K u n z , 1980). I m m u n i z a t i o n s t u d i e s w i t h c h e m i c a l l y defined v i r a l components revealed the glycoprotein as the major and most likely single compo-

T i c k - b o r n e e n c e p h a l i t i s ( T B E ) v i r u s is o n e o f t h e m o s t h i g h l y p a t h o g e n i c flaviv i r u s e s f o r m a n ; i t i s e n d e m i c in s e v e r a l European countries, Russia, and probably China. In Europe it represents by far the most important arthropod-transmitted d i s e a s e a n d in c e r t a i n c o u n t r i e s i t is o f major public health interest because of the hundreds of cases hospitalized each year. A vaccine has been developed containing highly purified inactivated virus (Heinz et 1To whom reprint requests should be addressed. 2Present address: Institute for Virus Research, German Cancer Research Centre, D-6900 Heidelberg, Germany. 525

0042-6822/83 $3.00 Copyright 9 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

526

H E I N Z ET AL.

nent inducing neutralizing antibodies and protective immunity (Heinz et al., 1981). Based on a detailed radioimmunological antigenic analysis of purified glycoproteins from members of the Murray Valley encephalitis subgroup and dengue viruses, Trent (1977) presented evidence for a complex antigenic structure of the viral glycoprotein; this structure is compatible with the presence of subtype- and type-specific, complex-reactive and flavivirus group-reactive antigenic determinants. These antigenic specificities form the basis for the serological classification of flaviviruses, as determined by haemagglutination-inhibition (HI) and neutralization tests; the classification system includes division into subgroups or complexes, types, subtypes, and eventually strains (for a review see Porterfield, 1980). The so-called TBE complex of flaviviruses is formed by TBE virus together with Langat, Negishi, Omsk haemorrhagic fever, Kyasanur Forest disease, and Louping ill viruses (De Madrid and Porterfield, 1974), all of which are very closely related and difficult to differentiate. Based on antibody absorption experiments (Clarke, 1964), TBE virus strains could be further assigned to a western (European) subtype which is primarily transmitted by Ixodes ricinus and a Far Eastern subtype with Ixodes persulcatus as its main vector. This subtype differentiation was confirmed by competitive RIA and peptide mapping using limited proteolysis of the corresponding structural glycoproteins (Heinz and Kunz, 1981). The observed serological heterogeneity reflects the potential for sequence or conformational changes within immunogenic regions of the viral glycoprotein, changes which would not impair primary protein functions in cell infection. Similar to the membrane glycoproteins of other enveloped viruses, the flavivirus E protein therefore is a strongly variable protein at least at certain antigenically active domains. One approach to the study of structural and functional identification of antigenic determinants involves the application of monoclonal antibodies as specific reagents

for defining single epitopes on the complex antigenic structure of protein molecules. Using this technology we have established a model for the antigenic structure of a flavivirus glycoprotein and have showed the distribution and function of conserved and variable epitopes. MATERIALS AND METHODS

Virus strains. Isolates of TBE virus analysed in the present study are listed in Table 1. Other flaviviruses analysed were the Far Eastern subtype of TBE virus (strain Sofyn), Louping ill (LI) virus (strain Moredun: Scotland), West Nile (WN) virus, and Murray Valley encephalitis (MVE) virus (prototypes of Yale Arbovirus Research Unit, New Haven, Conn.). Investigators who had kindly provided some of these viruses are fully acknowledged in a previous publication (Heinz and Kunz, 1981). Propagation and purification of viruses. Suspensions of infected suckling mouse brain were used to infect primary chick embryo cells which were maintained in minimum essential medium (MEM) buffered with 15 m M H E P E S and 15 m M E P P S at pH 7.6. Supernatants were harvested at 40 hr p.i., clarified, and subjected to ultracentrifugation to pellet virus which was then resuspended and further purified by rate zonal followed by equilibrium density gradient centrifugation. Experimental details of procedures have been described by Heinz and Kunz (1981). Preparation of glycoprotein complexes and cores. Glycoprotein complexes obtained after Triton (TX)-100 and cetyltrimethylammonium bromide (CTAB) solubilization were prepared as described by Heinz and Kunz (1980). Briefly, detergent was added to a suspension of purified virus at a detergent-to-protein ratio of 10:1 (TX100) or 5:1 (CTAB), allowed to stand for 1 hr at room temperature, and then subjected to density gradient centrifugation in detergent-free sucrose gradients. This resulted in the formation of defined polymeric glycoprotein complexes. These contained both envelope proteins (E and M) after TX-100 solubilization, whereas E was

ANTIGENIC STRUCTURE OF TBE VIRUS GLYCOPROTEIN

527

TABLE 1 STRAINS OF TBE VIRUSESa (WESTERN SUBTYPE) ANALYSED Geographic origin

Strain designation

Year of isolation

Source of isolation

Austria

Neud5rfl St. PSlten Hochosterwitz Scharl

1971 1971 1971 1956

Ixodes ricinus Ixodes ricinus Ixodes ricinus H u m a n brain

West Germany

K-23

1975

Ixodes ricinus

Switzerland

1-40 I-Horse

1972 1979

Ixodes ricinus Horse brain

Finland

A-52 S-10-12

1959 1960

Ixodes ricinus Ixodes ricinus

Czechoslovakia

Hypr

1953

H u m a n blood

These s t r a i n s are highly pathogenic and therefore cannot be made available for general distribution. The editors have agreed to publish this paper despite t h a t restriction.

the only protein constituent after CTAB solubilization. Cores were recovered from the density gradient pellets after TX-100 solubilization. The purity of the different preparations was ascertained by SDSP A G E in the buffer system of Laemmli and Favre (1973) and protein concerttrations were determined according to Schaffner and Weissmann (1973).

Preparation of monoclonal antibodies. BALB/e mice 2 months of age were immunized i.p. with glycoprotein complexes derived from TBE virus (strain Neud5rfl) after solubilization with TX-100. The first dose (5 #g) was emulsified in complete Freund's adjuvant and three f u r t h e r boosting doses without adjuvant were given 1, 3, and 4 weeks apart. Fusion of spleen cells with mouse myeloma cells was performed essentially as described by KShler and Milstein (1975). Briefly, PEG 4000 at a final concentration of 50% (w/v) was used to fuse 1 X 10s spleen cells with 1 X 107 mouse myeloma cells (X63-Ag8/ 653) which were then suspended in HAT medium (50 ml) and seeded into a total of eight microtiter tissue culture plates (100 td/well). Supernatants from individual wells were assayed for production of specific antibodies in an enzyme immunoassay using polystyrene microtitre plates coated with purified virus as a solid phase

(see below). Cultures of hybridomas selected for cloning were subjected to limiting dilution using BALB/c thymocytes as feeder cells. Production of immune ascitic fluid. One week after priming adult BALB/c mice with pristane (2,6,10,14-tetramethylpentadecane; EGA Chemie, Steinheim, FRG), 1 X 107 viable hybridoma cells were injected i.p. Ascitic fluids were recovered, clarified and stored at - 2 0 ~. Enzyme immunoassay (EIA). Polystyrene microtitre plates (Nunc, Kamstrup, Denmark) were coated by overnight incubation with purified virus, glycoprotein complexes, or cores at a protein concentration of 2 tLg/ml in carbonate buffer, pH 9.6. The solid phase was absorbed by incubation for 1 hr at 37 ~ with phosphatebuffered saline (PBS), pH 7.4, containing 2% sheep serum, then the plates were emptied and the test samples were allowed to react for 1 hr at 37 ~ Hybridoma supe r nat ant s were tested undiluted; titrations of immune ascitic fluids were performed in PBS, pH 7.4, containing 2% sheep serum. After sample incubation the wells were emptied and washed three times with PBS (pH 7.4) and incubated with horseradish peroxidase-conjugated rabbit-anti-mouse Ig (Nordic, Biogenzia Lemania SA, Switzerland) in PBS (pH 7.4)

528

H E I N Z ET AL.

containing 2% sheep serum for I hr at 37 ~ After three washes with PBS, substrate (o-phenylenediamine, 1 m g/ m l in phosp h a t e - c i t r a t e buffer, pH 5.0, plus 1 #1 perhydrol/ml) was added and the enzyme reaction was stopped after 30 rain at room t e m p e r a t u r e by the addition of 2 N H2S04. The absorbance at 492 nm was measured with a multichannel photometer (Multiskan, Flow Laboratories, Bonn, Germany). Determination of antibody class and subclass. For this purpose the enzyme immunoassay described above was modified by the introduction of a second layer of subclass-specific rabbit sera against mouse IgG, IgM, IgG1, IgG2ab, IgG2a and IgG3 (Nordic, Biogenzia Lemania SA, Switzerland) which were then detected by goatanti-rabbit Ig serum conjugated with horseradish peroxidase. Haemagglutination inhibition test. Haemagglutination-inhibition assays were performed as described by Clarke and Casals (1958). Assays were performed at pH 6.4 using goose erythrocytes. Isolation and 125I-labelling of monoclonal antibodies. Antibodies present in mouse ascitic fluids were precipitated with ammonium sulfate at 50% saturation in PBS (pH 7.4). The precipitate was washed once in 50% saturated ammonium sulfate and then resuspended in PBS pH 7.4. This preparation was desalted by gel filtration using a PD-10 column (Pharmacia, Uppsata, Sweden) equilibrated in PBS (pH 7.4). Iodination using Iodogen R (Pierce Eurochemie, Rotterdam, Holland) was carried out as described by Markwell and Fox (1978). Glass tubes were coated with 2.5 #g Iodogen in 100 #1 of chloroform, which was removed by a gentle stream of nitrogen. Ammonium sulfate-precipitated immunoglobulin (25 #g in 100 #1) was then added together with 300 ttCi [125I]Na (Amersham International), allowed to react for 15 rain in an ice bath, and then subjected to gelfiltration in a PD-10 column (Pharmacia, Uppsala, Sweden). Antibody blocking assay. Polystyrene beads (6.4-mm diameter, Precision Ball Co., Chicago, Ill.) were coated with purified TBE virus (2 ttg/ml in carbonate buffer, pH 9.6) by overnight incubation at 4 ~. Before the

beginning of the test, coated beads were absorbed by incubation for I hr at 37 ~ with PBS, pH 7.4, containing 2% sheep serum. Single beads were then added to polystyrene tubes and all f u r t h e r operations were carried out in a volume of 0.2 ml/bead. Ascitic fluids containing monoclonal antibodies diluted from 10-1 to 10-6 in PBS (pH 7.4) containing 2% sheep serum were added to block the corresponding epitope on the virus. After 1 hr at 37 ~ the beads were washed four times with tap water. A fixed amount of 12~I-labelled monoclonal antibody in the same buffer as above was added and incubated for 1 hr at 37 ~ The beads were again washed four times with tap water and counted in a ~,-spectrometer. Passive mouse protection test. Groups of 10 mice each were passively immunized with fourfold dilutions (1:4 to 1:256) of ascitic fluids containing monoclonal antibodies. Each mouse received a dose of 0.2 ml subcutaneously and was then challenged 24 hr later by intraperitoneal inoculation of 100 to 500 LD~0 of TBE virus. The observation period was 18 days, and results are expressed as titer, i.e., the reciprocal of t h a t dilution which protects 50% of the immunized mice. Neutralization test. Dilutions of ascitic fluids (1:10, 30, 100, 300, 1000, 3000, 10000) in TCM containing 10% fetal calf serum were mixed with an equal volume containing 100 TCID~0 of TBE virus. After an incubation period of 90 min at 37 ~ PScells were added to yield a final concentration of 1 X 106 cells/ml. One hundred microliters of these mixtures were seeded into the wells of flat-bottomed microtitre tissue culture plates (Nunc, Kamstrup, Denmark) and incubated at 37 ~ Each ascitic fluid dilution was tested in triplicate and results Were read after 4 days. RESULTS The strategy of our investigation was focused on the selection of monoclonal antibodies specific for different antigenic sites on the structural glycoprotein (E) of TBE virus. This goal was achieved by first isolating a panel of antibodies reactive with

ANTIGENIC STRUCTURE OF TBE VIRUS GLYCOPROTEIN TABLE 2 MONOCLONALANTIBODIESAGAINST TBE VIRUSE PROTEIN Monoclonal antibody 1C4 6E2 1B3 2B6 4D9 1G2 2E7 5D6

Antibody class/ subclass

Titer in EIA a

IgG1 IgG2a IgG1 IgG1 IgG1 IgG1 IgG1 IgG1

1 • 106 3 x 106 1 • 10G 1 • 10~ 1 • 10G 2 X 107 5 X l0 G 5 X 10~

Reciprocal of that dilution, at which titration curve crosses cutoff line (0.1 absorbance units).

t h e g l y c o p r o t e i n in e n z y m e i m m u n o a s s a y a n d t h e n a p p l y i n g v a r i o u s t e s t s to s e l e c t such a n t i b o d i e s which differed in a t l e a s t one o p e r a t i o n a l or f u n c t i o n a l c r i t e r i o n . This a p p r o a c h defined u n i q u e e p i t o p e s on t h e g l y c o p r o t e i n . The t e s t s y s t e m s u s e d w e r e v a r i a n t a n a l y s i s , H I test, n e u t r a l i z a tion test, passive mouse p r o t e c t i o n test, a n d m u t u a l b l o c k i n g t e s t s f o r t h e a n a l y s i s of spatial relationships between different anTBE Virus

Rosettes (TX-IOO)

529

tibodies. By t h i s m e a n s e i g h t m o n o c l o n a l antibodies were selected which reacted w i t h e i g h t d i s t i n g u i s h a b l e e p i t o p e s . These e i g h t a n t i b o d i e s a r e d e s c r i b e d in d e t a i l in t h e p r e s e n t r e p o r t a n d a r e l i s t e d in T a b l e 2 t o g e t h e r w i t h t h e i r s u b c l a s s a n d t i t e r of t h e c o r r e s p o n d i n g a s c i t i c fluid in E I A . A s can be seen, all a n t i b o d i e s a r e of IgG1 subclass e x c e p t one ( d e s i g n a t e d 6E2) which is IgG2a and therefore protein A-binding. The a n t i g l y c o p r o t e i n specificity of t h e m o n o c l o n a l a n t i b o d i e s w a s verified in an e n z y m e i m m u n o a s s a y u s i n g pure, defined v i r a l c o m p o n e n t s . The i s o l a t i o n a n d imm u n o l o g i c a l c h a r a c t e r i z a t i o n of t h e s e p r e p a r a t i o n s h a v e been d e s c r i b e d in p r e vious p u b l i c a t i o n s ( H e i n z a n d Kunz, 1980; H e i n z et al., 1981) a n d t h e i r p o l y p e p t i d e c o m p o s i t i o n is s h o w n in Fig. 1. Thus, comp l e t e v i r u s c o n t a i n s t h r e e p o l y p e p t i d e s (E, C, a n d M); g l y c o p r o t e i n c o m p l e x e s obt a i n e d a f t e r TX-100 s o l u b i l i z a t i o n a r e c o m p o s e d of E a n d M, w h e r e a s t h o s e obt a i n e d a f t e r CTAB s o l u b i l i z a t i o n c o n t a i n E exclusively. C is the o n l y core protein~ Enzyme immunoassays with these prepa r a t i o n s r e v e a l e d t h a t all m o n o c l o n a l ant i b o d i e s b o u n d to p l a t e s c o a t e d w i t h comp l e t e v i r u s a n d w i t h g l y c o p r o t e i n comRosettes (CTAB)

Core

FIG. 1. SDS-PAGE of purified TBE virus, cores isolated after TX-100 treatment, and glycoprotein complexes ("rosettes") obtained after TX-100 and CTAB solubilization. Protein bands were stained with Coomassie brillant blue.

530

H E I N Z E T AL. Complete Virus 1

2

3

4

5

6

7

Core 8

9

1

2

3

4

5

6

7

8

9

-4

g'-5

-

~'

-6

;-7

E=

m r

m

GP-Complexes

T x-100 1

2

3

4

5

6

7

8

CTAB 9

1

2

3

GP-Complexes 4

5

6

7

8

9

0

FIG. 2. T i t r a t i o n of monoclonal antibodies a n d polyvalent m o u s e i m m u n e s e r u m in e n z y m e imm u n o a s s a y u s i n g m i c r o t i t r e plates coated w i t h purified virus, purified cores, a n d glycoprotein complexes obtained a f t e r solubilization with TX-100 a n d CTAB. 1 to 6: Monoclonal a n t i b o d i e s 1C4, 6E2, 1B3, 2B6, 4D9, 1G2; 7: n e g a t i v e ascites; 8: n e g a t i v e m o u s e s e r u m ; 9: m o u s e i m m u n e s e r u m .

plexes obtained after TX-100 as well as CTAB solubilization. These monoclonal antibodies did not bind to the nucleocapsid (Fig. 2). This indicates t h a t all of these antibodies recognize determinants on the glycoprotein in its native conformation in the viral envelope. These same determinants are also present on glycoprotein complexes obtained under mild solubilization conditions using either TX-100 or CTAB. The same result was obtained for antibodies 5D6 and 2E7 which are not included in Fig. 2.

Variant Analysis The complexity of the antigenic structure of the flavivirus glycoprotein is indicated by the evidence for group-, complex-, type-, and subtype-specific determinants (Clarke, 1960; Trent, 1977). For the precise determination of cross reactivities of monoclonal antibodies, we employed variant analysis in EIA using the homologous virus (TBE virus, Western subtype), the Far E a s t e r n subtype, another member of the TBE complex (LI virus), and two members of a more distantly related serocomplex (WN and MVE virus) as antigens. Purified virus preparations at

standardized concentrations were used to coat the solid phase in order that differences in absorbance values reflected true antigenic differences and not merely differences in antigen preparations. The antibody titration curves established (Fig. 3) represent characteristic serological fingerprints for each monoclonal antibody. These also reveal partial crossreactivities with single antigenic determinants caused by slightly changed epitopes. As can be seen from Fig. 3, the flavivirus group-reactive determinant defined by 6E2 is indistinguishably present on all viruses analysed. 2B6 defines a second group-reactive d e t e r m i n a n t which however, is altered on the two viruses from another subgroup (WN and MVE). Such partial cross-reactivities involving single determinants were also found for antibodies 1C4, 4D9, and 1G2. Interestingly, 1C4 and 4D9 allow the discrimination between Western and Far Eastern subtype of TBE virus by reacting with an epitope that is changed on the Far E a s t e r n subtype but which is present identically on LI virus. Although 1C4 and 4D9, as well as 1B3, 2E7, and 5D6, have similar serological fingerprints, they are functionally or topologically distinguishable (see next sections)

ANTIGENIC STRUCTURE OF TBE VIRUS GLYCOPROTEIN

2.0- .L

~

o~,

1.5- 9

1C4 ~ ~f

1.00.5

o:

TBE strains

~

6E2

strai~~ ns

LI WN

MVE i

1

1.5-

1B3

~, TBEstrains 9~

~

,11,

2 B6

kl

Sofyn

1.0"

E

i M W ~ N ~ ~ E i ~ I ~ ~'I I t

rails

",

MVE WN

~0.5-

o

~

TBE Sofyn

LI

o:" --1.

2.0-

\

~.

531

oc

m ~2.0- i

.i,

,i,

O (R J:

< 1.5- 9~ .

1,

4D9

\\

~ TBE strains ~ , LI

Sofyn ~

,~.

~

,L

1G2 TBE strains \ \Sofyn

"LI~,

1.O 0.5

WN r~

_1_

2D.

_1

~

2E7 ~TBE strains

.~.

~,

~;

~

~

~

~

0.5

MVE

MVE

L ~

WN 10 -2

,~.~.

5D6 I TBE-'~-rains

1.5 1.0

~

LI~~,~

WN 10 "3

10 .4

10"5

10"6

10 -7

10-2 10-3 10-4 10-5 10-6 10-7

Dilution of Ascites FIG. 3. Titration curves obtained in enzyme immunoassay with monoclonal antibodies (1C4, 6E2, 1B3, 2B6, 4D9, 1G2, 2E7, 5D6) using microtitre plates coated with purified preparations of TBE virus strains (Western subtype) (0), the F a r Eastern subtype (Sofyn) (A) louping ill (LI) virus (11), West Nile (WN) virus (9 and Murray valley encephalitis (MVE) virus (A) at a concentration of 2 #g/ml.

and t h e r e f o r e r e a c t w i t h different epitopes. In the a s s a y s s h o w n in Fig. 3, we h a v e also included all the w e s t e r n T B E vi-

rus s t r a i n s listed in T a b l e I to see w h e t h e r s u b t y p e v a r i a t i o n s a t t h e eight epitopes occur b e t w e e n E u r o p e a n isolates. Identi-

532

H E I N Z ET AL.

cal titration curves were obtained with all these strains and for the sake of clarity only one collective curve has been drawn.

Functional Tests The role of monoclonal antibody-defined epitopes in haemagglutination, neutralization, and protection from disease was analysed and the results are presented in Table 3. The binding of 1C4 and 1G2 did not inhibit haemagglutination; this was definitely not only a m a t t e r of antibody titer (Table 1) but reflected a true functional difference from the other HA-inhibiting antibodies. Haemagglutination inhibition tests with polyvalent immune sera usually reveal broad cross-reactivities between all flaviviruses, such as is exemplified in the upper part of Table 4 by cross HI results obtained with mouse immune sera. From their different specificities in the binding assay (Fig. 3), HA-inhibiting monoclonal antibodies (6E2, 2B6, 4D9, 1B3, 2E7, and 5D6) were also expected to reveal different degrees of cross-reactivities in HI tests and this is exactly what we found (Table 4, lower part). Antibody 6E2 is broadly group reactive, but, in cont ra s t to EIA, surprisingly gives much higher titres with WN and MVE virus than with the homologous antigen. 2B6, which TABLE 3 RESULTS OF HAEMAGGLUTINATION INHIBITION (HI), NEUTRALIZATION, AND PASSIVE MOUSE PROTECTION TESTS OBTAINED WITH MONOCLONAL ANTIBODIES AGAINST TBE VIRUS E PROTEIN T i t e r in

Monoclonal antibody

HI test

Neutralization test

P a s s i v e mouse protection test

1C4 6E2 1B3 2B6 4D9 1G2 2E7 5D6

<10 1280 20 320 640 <10 160 80/160

<10 <10 10 <10 100 <10 100 100

<4 a <4 4 <4 90 <4 62 15

a Less t h a n fo ur s u r v i v o r s a t a d i l u t i o n of 1:4.

compared to TBE viruses shows reduced binding to WN and MVE virus in EIA (Fig. 3) still gave identical titres in HI. Antibodies 1B3, 4D9, 2E7, and 5D6, however, reacted exclusively with TBE and LI viruses and thus define TBE complex-specific determinants involved in HI. None of the HI-negative antibodies revealed virus neutralization. That the latter is not necessesarily linked to HI activity is exemplified by the flavivirus groupreactive antibodies 6E2 and 2B6. Three TBE complex-reactive antibodies (1B3, 2E7, 5D6) as well as one subtype-specific antibody (4D9) did show neutralizing activity and also protected mice after passive immunization against a lethal challenge with TBE virus (Table 3).

Topography of Epitopes The topological relationship of the eight monoclonal antibody-defined epitopes was analysed in reciprocal blocking assays by determining which of the eight monoclonal antibodies was able to block the binding of each of the other antibodies (Fig. 4). It has to be kept in mind t h a t the "blocking" effects observed in such assays may be caused by different mechanisms, i.e., steric hinderance by binding to identical, overlapping, or closely adjacent epitopes on the one hand, or on the other hand induction of conformational changes. As can be seen in Fig. 4, the blocking of antibody pairs was always symmetrical and no one-way reactions were observed. Differences in the degree of blocking efficiency between antibody pairs were seen, however (e.g., antibodies 5D6 and 2E7 (Fig. 4)). 2E7 was blocked completely by 5D6, but 5D6 was blocked to only about 50% by the highest concentration of 2E7. With the exception of 1C4, each epitope was linked to at least one second epitope as revealed by mutual blocking of the corresponding antibodies. Based on the experiments presented above we have established a model showing the distribution, function, and serological specificities of eight distinct epitopes on the structural glycoprotein of TBE virus (Fig. 5). Although five independent antigenic determinants were defined by the

ANTIGENIC STRUCTURE OF TBE VIRUS GLYCOPROTEIN

533

TABLE 4 CROSSREACTIVITY OF MOUSE IMMUNE SERA AND MONOCLONAL ANTIBODIES IN H I TEST Test-Antigens

TBE virus

Mouse i m m u n e s e r a NeudSrfl L o u p i n g ill MVE Monoclonal a n t i b o d i e s 1C4 6E2 1B3 2B6 4D9 1G2 2E7 5D6

Western subtype (NeudSrfl)

Far Eastern subtype

160 80 20

160 80 20

<10 1280 20 320 640 <10 160 80/160

<10 1280 10 320 320 <10 160 80/160

monoclonal antibodies 1C4, 6E2, 4D9, 1B3 (5D6), and 1G2, seven of the eight antibodies were linked by m u t u a l blocking and t h e r e f o r e defined two m a j o r domains. As shown in detail in the figure both of these domains are h e t e r o g e n e o u s and contain epitopes which differ with respect to t h e i r involvement in HA, neutralization, and passive protection, as well as t h e i r degree of cross-reactivity. DISCUSSION P r o t e i n antigens m a y c a r r y a large n u m b e r of different antigenically reactive sites, and it has been suggested by Todd et al. (1982) t h a t even the whole surface of a protein (myoglobin) m a y r e p r e s e n t a contiguous antigenic area. However, as pointed out by A r n o n (1980), only a limited n u m b e r of these p o t e n t i a l l y antigenic sites seem to be " i m m u n o d o m i n a n t " and can p r e f e r e n t i a l l y s t i m u l a t e a host's i m m u n e response. In accord with this view, evidence has been p r e s e n t e d t h a t most of the a n t i b o d y response in an immunized host (as investigated with p o l y v a l e n t i m m u n e sera) is directed against relatively few antigenic sites and t h a t the antigenic dominance of these sites r e p r e s e n t s an inher-

Louping ill virus 160 160 20

< 10 1280 <10 320 640 <10 80/160 80

West Nile virus

Murray Valley Enc. virus

20/40 20/40 80

40 20/40 160

<10 10240 <10 320 <10 <10 <10 <10

<10 10240 <10 320 <10 <10 <10 <10

ent p r o p e r t y of t h e i r location in the threedimensional s t r u c t u r e of the protein (Atassi, 1980). As revealed by cross-reactivities in H I (Clarke, 1960), n e u t r a l i z a t i o n tests (DeMadrid and Porterfield, 1974), or r a d i o i m m u n o a s s a y s (Trent, 1977), using polyvalent i m m u n e sera, several serologically and p r o b a b l y also functionally distinguishable i m m u n o g e n i c d e t e r m i n a n t s seem to be p r e s e n t on the s t r u c t u r a l glycoprotein of flaviviruses. By the use of monoclonal antibodies, we were able to dissect the complex antigenic s t r u c t u r e of a flavivirus glycoprotein at the level of single antigenic d e t e r m i n a n t s . This allowed us to establish a map of eight distinct subtype-, type-, complex-, and group-specific epitopes on this glycoprotein which because of t h e i r different functions and serological specificities can explain m a n y of the earlier findings obtained with polyvalent i m m u n e sera. The topological m a p p i n g of these epitopes has revealed five i n d e p e n d e n t nonoverlapping antigenic sites, but also a clustering of most epitopes into two complex antigenic domains. C h a r a c t e r i s t i c a l l y these domains are inhomogeneous with respect to serological specificity as well as

534

HEINZ ET AL. 125 I - 1 C 4

1251 - 6 E 2

O.

/

20 40

/

6080-

//"

,-o 1 ~

.., 6 E 2 2B6

9

10o 1251-1B 3

1251-2B 6

0 20 4 0 84

60 o~ 80 c

,~

:-',11./

/

.-, 5DS

9/"

"~100 0 m

1251- 4D9

125 I- 1G2

0 o

o

~

20

"

40

2B6

60

:<~/~ o_o1 G 2 9-. 2E7

6/

8 0 84 100

o-o ?/'/ o/" --- 4 D 9

;s: 125

0 84

I

9

-2E7

1251

o/O;r

20 40

/ / / z / . ,~

5D6

-

- ~o - ~o - ~

- ~o~/~

/i/

,-.2 7

9-. 1 B 3

~-"2E7

60 e~=

/

10-1

10"3

80 100 10-1

10-3

10"5

.10-7

Dilution

10-5

10-7

of Blocking

Antibodies

FIG. 4. Mutual blocking assays between different monoclonal antibodies. The blocking of each of the eight distinct l~I-labelled monoclonal antibodies by the same but unlabelled antibodies was analysed by first incubating TBE virus-coated polystyrene beads with a dilution series of unlabelled antibodies and then with a fixed amount of 125I-labelled monoclonal antibody. f u n c t i o n . T h e e f f e c t of a n t i b o d y b i n d i n g (e.g., n e u t r a l i z a t i o n o r h a e m a g g l u t i n a t i o n i n h i b i t i o n ) s e e m s to d e p e n d on t h e e x a c t l o c a t i o n o f e a c h e p i t o p e in t h e t h r e e - d i -

mensional structure of the protein. As can b e s e e n in t h e m o d e l ( F i g . 5), H A - i n h i b i t i n g m o n o c l o n a l a n t i b o d i e s define g r o u p specific a s w e l l a s c o m p l e x - a n d s u b t y p e -

ANTIGENIC STRUCTURE OF TBE VIRUS GLYCOPROTEIN

535

HA- inhibiting A

neut ralizing .protective /~

groupspecific

HA- inhibiting r

- -

subtypespecific

partially group-specific 2B6

6E2

,

~

TBE - c o m p l e x specific

\

-

J

4D9

1B3

subtype specific

-

neutralizing + protective

5D6

2E7

type specific 1G2

\/

1C4

Structural of

TBE

glycoprotein virus

| I

I ,

FIG. 5. Topological and functional model of antigenic sites on the structural glycoprotein of TBE virus, as defined by monoclonal antibodies. Mutual blocking of antibodies is indicated by overlapping symbols in the figure.

specific epitopes. This is exactly what had to be postulated from the classical studies of Clarke (1960, 1964) who demonstrated t h a t broadly cross-reactive HI-reactions with polyvalent immune sera against flaviviruses can be made complex-, type-, or subtype-specific by absorption with appropriate heterologous antigens. The titration curves of antibody 6E2 obtained in EIA with flaviviruses of a different serogroup (WN, MVE) were indistinguishable from those seen with TBE virus. This indicates th a t the corresponding epitope is identical on the glycoproteins of these viruses and therefore represents a group-specific determinant. Interestingly, however, titres in HI-tests were much higher in the heterologous than in the homologous system; this indicates t ha t the HA activity of WN and MVE virus seems to be more sensitive to antibody binding

at this special epitope and thus represents an intrinsic property of the glycoproteins involved. The involvement of such epitopes may explain the well-known fact seen in animal studies with flaviviruses, as well as alphaviruses, t hat after consecutive inoculation with different viruses, heterologous HI-titres may well exceed those found against either of the inoculated viruses (reviewed by Porterfield, 1980). Flavivirus group-reactive monoclonal antibodies, although reactive in HI, were not involved in neutralization. This finding is compatible with the observation t h a t neutralization tests performed with polyvalent immune sera show higher serological specificity than HI-tests. Molecular epidemiological studies of European TBE virus isolates using peptide mapping of structural and nonstructural proteins as well as competitive RIA (Heinz

536

HEINZ ET AL.

and Kunz, 1981; Heinz and Kunz, 1982), have revealed a remarkable uniformity of this virus. This is now confirmed for the eight individual epitopes described in this study which are all identically present on strains isolated in different years from different species in Austria, Germany, Switzerland, Czechoslovakia, and Finland. Four out of the eight epitopes described do not seem to be involved in neutralization or protection. Nevertheless they may be of considerable importance and they may influence the impact of the overall immune response on the disease. In this context the observation t h a t antibodies can m e d i ate enhancement of infectivity is especially interesting. This phenomenon is dependent upon Fc-receptor functions of the host cell and has been most thoroughly studied in the flavivirus system (Halstead and O'Rourke, 1977; Halstead et al., 1980; Peiris and Porterfield, 1979) by demonstrating th at subneutralizing concentrations of polyvalent immune sera may enhance virus infectivity. In a recent study using monoclonal antibodies against WN virus, Peiris et al. (1982) have shown t hat antibody mediated enhancement of infectivity cannot only be caused by monoclonal antibodies involved in neutralization and HI but also by a subgroup-specific antibody with poor neutralizing and poor HI activity which is similar to the specifications of 1C4, 1B3, and 1G2 in the TBE virus system. Furthermore, antibodies t h a t do not neutralize in vitro may nevertheless be protective in vivo as has recently been demonstrated by Schmaljohn et al. (1982) for monoclonal antibodies against Sindbis virus. As suggested by these authors, the underlying mechanism could be complement-dependent lysis of virus-infected cells which can be mediated by nonneutralizing antibodies. From the monoclonal antibodies described in the present report, only those which neutralized the virus in vitro also passively protected mice against lethal challenge with TBE virus. A similar phenomenon may nevertheless also play a role in TBE virus infections since all the epitopes described in the present r e por t (with the exception of 6E2) are defined by antibodies of IgG1 subclass, which do not

mediate immunospecific complement-dependent cytolysis (Spiegelberg, 1974). To our knowledge, however, there is presently no report on the early expression of TBE virus E protein on the surface of infected cells which would allow antibody-mediated lysis. Special consideration has to be made for antibodies 2B6 and 1G2 which both are nonneutralizing and nonprotective but are capable of blocking the binding of neutralizing antibodies. A strong immune response against such determinants may well have important implications for the development and the outcome of disease. A similar blocking of neutralizing antibodies has also been described for monoclonal antibodies against mouse m a m m a r y tumour virus by Massey and Schochetman (1981), and it will be of great importance to investigate the in vivo relevance of such in vitro findings in the TBE virus as well as other viral systems. It is probable t hat the relative amounts and affinities of antibodies elicited in the course of virus replication against critical epitopes (e.g., neutralizing, blocking, infection-enhancing) besides other factors may influence the outcome of virus infections manifested by different clinical pictures. Since the immune response to single antigenic determinants has been demonstrated to be under separate genetic control (Maron et aL, 1973; Mozes et al., 1971; Milich and Chisari, 1982) and the exact binding site of an antibody within a heterogenic antigenic domain seems to determine its function, it would be especially interesting to analyse the immune response of infected individuals on the basis of single epitopes. Although we cannot exclude that additional sites may be involved in the natural immune response to the TBE virus glycoprotein, our model can fully explain results obtained with polyclonal immune sera from different species and we therefore believe t h a t it accounts for a major portion of the protein's immunologically relevant domains. REFERENCES ARNON, R. (1980). Chemically defined antiviral vaccines. Annu. Rev. Microbiol. 34, 593-618. ATASSI, M. Z. (1980). Precise determination of protein

ANTIGENIC STRUCTURE OF TBE VIRUS GLYCOPROTEIN antigenic structures has unravelled the molecular immune recognition of proteins and providecl a prototype for synthetic mimicking of other protein binding sites. MoL CelL Biochem. 32, 21-43. CLARKE, D. H., and CASALS, J. (1958). Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Amer. J. Trop. MeeL Hyg. 7, 561-573. CLARKE, D. H. (1960). Antigenic analysis of certain group B arthropod-borne viruses by antibody absorption. J. Exp. Mec~ 111, 1-20. CLARKE,D. H. (1960). Antigenic analysis of certain group B arthropod-borne viruses by antibody absorption. J. Exp. Med 111, 1-20. DE MADRID, A. T., and PORTERFIELD, J. S. (1974). The flaviviruses (group B arboviruses): A cross-neutralization study. J. Gen. ViroL 23, 91-96. HALSTEAD, S. B., and O'RoURKE, E. J. (1977). Dengue viruses and mononuclear phagocytes. I. Infection enhancement by non-neutralizing antibody. J. Exp. Med. 146, 201-217. HALSTEAD, S. B., PORTERFIELD, J. S., and O'ROURKE, E. J. (1980). E n h a n c e m e n t of dengue virus infection in monocytes by flavivirus antisera. Amer. J. Trop. MecL Hyg. 29, 638-642. HEINZ, F. X., and KUNZ, CH. (1980). Formation of polymeric glycoprotein complexes from a flaviviruN: Tick-borne encephalitis virus. J. Gen. Virol. 49, 125-132. HEINZ, F. X., KUNZ, CH., and FAUMA, H. (1980). Preparation of a highly purified vaccine against tickborne encephalitis virus by continuous-flow zonal ultracentrifugation. J. Me(L Virol. 6, 213-222. HEINZ, F. X., TUMA, W., and KUNZ, CH. (1981). Antigenic and immunogenic properties of defined physical forms of tick-borne encephalitis virus structural proteins. Infect. Immun. 33, 250-257. HEINZ, F. X., and KUNZ, CH. (1981). Homogeneity of the structural glycoprotein from European isolates of tick-borne encephalitis virus: Comparison with other flaviviruses. J. Gen. ViroL 57, 263-274. HEINZ, F. X., and KUNZ, CH. (1982). Molecular Epidemiology of tick-borne encephalitis virus: Peptide mapping os large nonstructura] proteins of European isolates and comparison with other flaviviruses. J. Gen. ViroL 62, 271-285. K()HLER, G., and MILSTEIN, C. (1975). Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature (London) 256, 495-497. KUNZ, CH., HEINZ, F. X., and HOFMANN,H. (1980a). Immunogenicity and reactogenicity of a highly purified vaccine against tick-borne encephalitis. J. Med. ViroL 6, 103-109. KUNZ, CH., HEINZ, F. X., and HOFMANN,H. (1980b). The efficacy of vaccination against tick-borne encephalitis. Wiener Klin. Wochenschr. 92, 809-813. LAEMMLI, U. K., and FAVRE, M. (1973). Maturation of

537

the head of bacteriophage T4. I. DNA packaging events. J. MoL Biol. 80, 575-599. MARKWELL, MARYANN K., and Fox, C. F. (1978). Surface specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3a,6~-diphenylglycoluril. Biochemistry 17, 4807-4817. MARON, E., SHER, H. I., MOZES, E., ARNON, R., and SELA, M. (1973). Genetic control of immune response toward the loop region of lysozyme. J. ImmunoL l l l , 101-105. MASSEY, R. J., and SCHOCHETMAN, G. (1981). Viral epitopes and monoclona] antibodies: isolation of blocking antibodies t h a t inhibit virus neutralization. Science 213, 447-449. MILICH, D. R., and CHISARI,F. V. (1982). Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). I-H-2 Restriction of the murrine humoral immune response to the a and d determinants of HBsAg. J. Immunol. 129, 320-325. MOZES, E., MARON, E., ARNON, R., and SELA, M. (1971). Strain dependent differences in the specificity of antibody responses toward lysozyme. J. ImmunoL 106, 862-864. PETRIS, J. S. M., and PORTERFIELD, J. S. (1979). Antibody-mediated enhancement of flavivirus replication in macrophage cell lines. Nature (London) 282. 509-511. PEIRIS, J. S. M., PORTERFIELD, J. S., and ROEHRIG, J. T. (1982). Monoclonal antibodies against the flavivirus West Nile. J. Gen. ViroL 58, 283-289. PORTERFIELD, Z. S. (1980). Antigenic characteristics and classification of togaviridae. In "The Togaviruses. Biology, Structure, Replication." (R. W. Schlesinger ed.), pp. 13-46. Academic Press, New York. SCHAFFNER, G., and WEISSMANN, C. (1973). A rapid, sensitive and specific method for the determination of protein in dilute solution. Anal Biochem. 56, 502514. SCHMAI.JOHN, i . L., JOHNSON, S. D., DALRYMPLE, J. M., and COLE, G. A. (1982). Non-neutralizing monoclonal antibodies can prevent lethal alphavirus encephalitis. Nature (London) 297, 70-72. SPIEGELBERG,H. L. (1974). Biological activities of immunoglobulins of different classes and subclasses. Adv. ImmunoL 19, 259-294. TODD, P. E. E., EAST, I. J., and LEACH, S. J. (1982). The immunogenicity and antigenicity of proteins. Trends in Biochem. Sc/. 7, 212-216. TRENT, D. W. (1977). Antigenic characterization of flavivirus structural proteins separated by isoelcctric focussing. J. ViroL 22, 608-618. WESTAWAY, E. G., SCHLESINGER, n. W., DALRYMPLE, J. M., and TRENT, D. W. (1980). Nomenclature of flavivirus-specified proteins. Intervirology 14, 114117.