1

Journal o f General Virology (1990), 71, 2065-2072.

Printed in Great Britain

2065

Mapping of antigenic sites on the bovine ephemeral fever virus glycoprotein using monoclonal antibodies D. H. Cybinski,* P. J. Walker, K. A. Byrne and H. Zakrzewski CSIRO Division of Tropical Animal Production, Long Pocket Laboratories, Private Bag No 3 PO, lndooroopilly, Queensland 4086, Australia

Monoclonal antibodies (MAbs) were produced against the G, M2 and N proteins of bovine ephemeral fever virus (BEFV) and 29 were selected for further study. Thirteen neutralizing MAbs were assigned to one conformation-independent and at least two conformation-dependent antigenic sites on the G protein by a competitive binding ELISA. The panel of MAbs were tested by neutralization and immunofluorescence with

three strains of BEFV and three BEFV-related viruses. The results indicated that BEFV strains from different sources were not identical and that the M2 protein was the least variable of the proteins investigated. Passive protection studies in mice showed that the correlation between neutralizing titre and resistance to challenge was 0-85 (P < 0.001).

Introduction

investigated. The cross-reactivity of neutralizing and non-neutralizing MAbs to three BEFV proteins was determined for three BEFV strains and three BEFVrelated viruses.

Bovine ephemeral fever virus (BEFV) is an arthropodborne rhabdovirus which causes an acute and disabling inflammatory disease in cattle and water buffalo. The BEF virion consists of five structural proteins, which have been designated L (180K), G (81K), N (52K), M1 (43K) and M2 (29K) (P. J. Walker et al., unpublished results). The G protein is an N-glycosylated membrane protein that forms projections on the virion surface and which can be removed by treatment with trypsin or nonionic detergent (Della-Porta & Brown, 1979; P. J. Walker et al., unpublished results). BEFV is structurally and serologically related to rabies virus (P. J. Walker et al., unpublished results; Calisher et al., 1989; Murphy et al., 1972) although the viruses differ in many properties including host range, tissue tropism and in the size of the G protein. In rabies virus, the G protein is the antigen which is thought to elicit and react with neutralizing antibody (Wiktor et al., 1973). As many as six antigenic sites have been defined on the rabies virus glycoprotein (G protein) using neutralizing monoclonal antibodies (MAbs) (Lafon et al., 1983; Wiktor et al., 1983; Bunschoten et al., 1989). MAbs have also been used to demonstrate antigenic variation in both the G and N proteins of rabies virus strains and rabies-related viruses (Flamand et al., 1980). In this study neutralizing MAbs were characterized and assigned to antigenic sites on the BEFV G protein on the basis of competitive binding assays and the role of neutralizing antibody in passive protection of mice was 0000=9456 © 1990 SGM

Methods Viruses and cells. The BB7721 strain of BEFV, isolated in mice from the blood of an infected cow (Doherty et al., 1969) was used for the production and testing of MAbs. The BEFV strains CSIRO 42 and CSIRO 53 were isolated in mice from mosquitoes (Standfast et al., 1976). Berrimah (BRM) virus strain DPP63, Kimberley (KIM) virus strain CSIRO 368 and Adelaide River (AR) virus strain DPP61 were isolated in BHK-21 tissue culture from healthy cattle (Gard et al., 1983, 1984). All viruses were propagated in BHK-21 cells and plaque-purified in Veto cells. Production of murine hybridomas. Two types of antigen were employed to generate BEFV antibodies in mice. The first was prepared from BEFV-infected suckling mouse brain, homogenized in a 10~ phosphate-buffered saline (PBS) suspension and centrifuged at 1000g; the supernatant was used as the inoculum. The second, purified BEF antigen, was prepared by a method to be described elsewhere, then treated with Triton X-100 to remove the G protein, centrifuged at 265 000 g, and the nucleocapsid pellet containing little or no G protein was used as the inoculum. Adult male BALB/c mice were injected intraperitoneally with 0-5 ml of a mixture of BEFV antigen and an equal volume of Freund's complete adjuvant. A similar inoculation was given 1 week later using Freund's incomplete adjuvant, followed by four intraperitoneal injections over the following 2 months using 0.5 ml of BEFV antigen and no adjuvant. The fusion was carried out 4 days after the final injection by a method based on that of K6hler & Milstein (1976). Fusion between BEFV-immune spleen cells and P3-X63-Ag8653 myeloma ceils (Kearney et aL, 1979) was induced with 50~ polyethylene glycol 1500 (w/v) in 75 mM-HEPES (Boehringer Mann-

2066

D. 11. Cybinski and others

helm). Mouse peritoneal macrophages served as feeder layers in 2496-well tissue culture plates. Supernatants were screened for BEFV specificity by using an immunofluorescence assay (IFA) and positive clones were subcloned by limiting dilution. High concentrations of IgG in ascites fluid were obtained by inoculating 106 to t07 hybrid cells intraperitoneally into BALB/c mice primed 1 to 3 weeks previously with 0.3 ml Pristane (2,6,10,14-tetramethylpentadecane) as described by Brodeur et al. (1984). Ascites fluids were clarified, heat-inactivated at 56 °C for 30 min and stored at - 2 0 °C.

Antibody testing. Hybridoma supernatants were screened using an indirect IFA which was described by Cybinski & Zakrzewski (1983), and modified for use in 96-well fiat-bottomed tissue culture plates. Monolayers of Vero cells were inoculated with 500 TCID5o of BEFV. After a 22 h incubation at 37 °C in an atmosphere of 5 ~ CO2, the plates were rinsed with 50% acetone in PBS and then fixed for 30 min with 100 ~tl/well of 7 0 ~ acetone in PBS. Hybridoma supernatants were incubated in the wells for 30 rain at 37 °C, washed with PBS, then stained with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin (Dako) for 30 min at 37 °C. After washing again with PBS the plates were examined by fluorescent microscopy. The presence of IFA-positive antibodies to other viruses was detected in a similar manner. Neutralization tests were carried out in Vero cells by a modification of the method described by Cybinski et al. (1978) using either undiluted hybridoma supernatant or ascites fluid in twofold dilutions in 96-weU microtitre plates and adding 100 TCIDso of test virus. Titres were expressed as the reciprocal dilution of ascites fluid that neutralized the virus in 50% of the wells. Polyclonal antibodies for use in crossneutralization tests were prepared in mice or rabbits as described by Sartorelli et al. (1966) and Cybinski & Zakrzewski (1983). Isotyping. Isotyping of antibodies was carried out using a mouse monoclonal isotyping kit (Commonwealth Serum Laboratories) which detects Ig heavy and light chains by means of a urease-labelled antiIfiouse Fab conjugate.

Purification of immunoglobulin. MAbs were purified by affinity chromatograpby of ascites fluid using DEAE-Affigel Blue (Bio-Rad) as described by Bruck et al. (1982). Fractions were analysed by SDSPAGE and IFA and those containing pure BEFV-specific IgG were pooled and concentrated by ultrafiltration using a Diaflo ultrafiltration membrane YM10 (Amicon), dialysed against 5 0 ~ glycerol in 20 m i Tris-HC1 and stored at - 2 0 °C. The concentration of IgG was estimated by a method described by Redinbaugh & Campbell (1985) using a Bio-Rad y-globulin standard. Competitive binding assay. Purified MAbs were dialysed against 100 mM-NaHCO3 pH 8.2 and diluted to approximately 1 mg/ml IgG. NHSLC biotin (Pierce Chemicals) in DMSO was added as described by Gretch et al. (1987) and allowed to react at room temperature for 2 h; then the products were dialysed extensively against 0.02~ NaN3 in PBS and stored at 4 °C. ELISA plates were coated overnight at 4 °C with gradient-purified BEFV antigen. All subsequent incubations were conducted at room temperature. Unbound sites were first blocked with 1~ gelatin and then the plates were incubated with 10-fold dilutions of one MAb which had been adjusted to 100 ~g/ml IgG, followed by the addition of biotinylated antibody at a predetermined dilution (giving an absorbance of between 0.4 and 0-6), and incubation for 1 h. Streptavidinbiotinylated horseradish peroxidase complex (Amersham), diluted 1000-fold was added and incubated for 1 h, followed by the addition of the substrate 5-amino salicylic acid with 0-002% H20 2. After 30 rain, the absorbance was measured in an ELISA reader (Titertek Multiskan M K l l ) using a 492 nm filter. The percentage competition was calculated using the formula [ 1 0 0 ( A - n ) ] / ( A - B ) , where A is the absorbance in the absence of competitor, B is the absorbance in the

presence of homologous antibody and n is the absorbance in the presence of competing antibody (Hughes et al., 1988). Reactions were graded as competition ( > 75 ~), partial competition (35 ~ to 75 %), noncompetition ( - 3 5 ~ to 34~) and enhancement (E) or negative competition ( < - 35%).

Mouse passive protection test. Litters of [ to 2 day old mice were inoculated intraperitoneally with 30 ~tl ascites fluid containing either neutralizing or non-neutralizing MAb. Control mice received no antibody. After 2 h, the mice were inoculated intracerebrally with 20 ~tl of 1000 5 0 ~ lethal doses of BEFV grown in mouse brain. The mice were examined daily for 14 days and deaths were recorded.

Results Production and characterization o f M A b s Using antigen 1 (BEFV-infected suckling mouse brain) as t h e i n o c u l u m , 26 s t a b l e , M A b - p r o d u c i n g h y b r i d o m a s were obtained after cloning. The antigenic specificity, i s o t y p e a n d v i r u s n e u t r a l i z i n g a b i l i t y o f e a c h c l o n e is s h o w n i n T a b l e 1. B y p r o t e i n i m m u n o b l o t t i n g ,

21 M A b s

T a b l e 1. General character&tics o f monoclonal antibodies

MAb

BEFV protein specificity by immunoblotting

Isotype*

DB5 13C6 13A3 9C5 17B 1 18D2 3A2 11B5 8B6 8D3 3D6 16A6 1C6 12A5 15B5 llD1 10BI 13B5 2C4 18C2 EB4 2C6 FD2 4A4 17A3 20A6

G G G G G G:~§ G G~§ G§ G§ G§ G§ G§ G§ G§ G§ G G:~§ G ~§ G G M2 M2 M2 M2 M2

IgG2a IgG2a IgG3 IgG2a IgG2a IgG2a IgG2b IgG2b IgG2a IgG2a IgG I IgG2a IgG1 IgG2a IgG 1 IgG1 IgG2a IgG2a IgG 1 IgG2a IgG2a IgG 1 IgG2a IgG2a IgG 1 IgG2a

11 A 3

N

IgG 1

6C1 1 C4

N

IgG2a

N

IgG 1

* All K light chains. t + , Neutralization; - , no neutralization. :~ Weak reaction. § Reaction only when 2-ME was absent.

BEFV neutralization'[" + + + + + + + + + + + + + -

Mapping of BEFV antigenic sites 120

reacted with the virion glycoprotein (G) and five reacted with the matrix protein (M2). Nine of the G-protein specific antibodies reacted in immunoblots after treatment with SDS and 2-mercaptoethanol (2-ME) whereas 12 others reacted only in the absence of 2-ME. Thirteen of the G protein-specific MAbs were capable of neutralizing BEFV in tissue culture. Using antigen 2 (BEFV nucleocapsids) as inoculum, 79 MAb-producing hybrids were detected by IFA and these were mainly specific for the nucleoprotein (N). Six of 10 cloned hybrids were stable and three of these were selected for further characterization (Table 1).

(a)

100 80 60 40 2O

ol -20

u

~

i

i

40 -

60 i

i

100

80

Competitive binding assays

60 40 20

~.

-20

~

-40

t

-

120

~'

~oo [

~

2067

8o 60 41)I 201 20 -40 -60 -80 -

- 100 - 120 /

I

r

I

110

~

~

~

loo - (d)

I

~

Thirteen neutralizing MAbs were biotinylated and reciprocal competitive binding assays were performed to determine the topological relationship of antigenic sites. Fig. 1 shows examples of the results of competition experiments using biotinylated MAbs DB5, 15B5, 11D1 and 17B1. In each case, antigen binding of biotinylated antibodies was blocked efficiently by homologous competing antibody. When competing heterologous antibody was in excess there was generally a 'plateau' at approximately 100~ or 0 ~ competition, although there were exceptions where only partial competition was observed. Examples of partial competition shown in Fig. 1 are llD1 and DB5 which partially inhibited the binding of biotinylated 15B5 and 17B1 respectively. Several antibodies were enhanced by the binding of other antibodies. Table 2 shows a summary of the competitive binding assays from data obtained using excess competing antibody. The results indicate that there are at least three

_

9O

T a b l e 2.

80, 70 60

~

G1

lol 0! ] -1

~

~

0

1

II~--

IgG competing antibody (log~0~g/ml)

Fig. 1. Competitive binding assay using a constant amount of biotinylated antibody and varying concentrations of competing antibodies. (a) M A b s DB5 ( 0 ) , 9C5 ( ~ ) , 16A6 ( . ) , l l D 1 (O), 8B6 ( A ) and 12A5 (IS])competing with biotinylated DB5; (b) M A b s DB5 (0),

17B1 ( A ) , 12A5 (m), 15B5 (O), l l D 1

( A ) and 8B6 (I--1)

competing with biotinylated 15B5; (c) M A b s 13C6 ( 0 ) , 17B1 ( A ) , 1C6 ( I l L 11D1 (O), 3D6 ( A ) and 16A6 ([S]) competing with biotinylated l l D 1 ; ( d ) M A b s DB5 (O), 17B1 ( A ) , 1C6 ( m ) , l i D 1 (O), 3D6 (/k) and 8B6 ([S]) competing with biotinylated 17B1.

BEFV glycoprotein

Biotin-labelled M A b * Antigenic site

40q ~ 30 I 20

--

on the

•

50

10

Antigenic sites

G2

G3a G3b

MAb 1 DB5 2 13C6 3 13A3 4 17Bl 5 9C5 6 1C6 7 12A5 8 15B5 9 IIDI 10 3D6 11 8B6 12 16A6 13 8D3

1

2

3

4

+ + + + ±+--± + + + + + ± + +

5

6

7

8

9

10 11 12 13 +

E

-

E

E

+ + +

+ + +

+ + +

+ + +

-

+

E

-

+ +

E E

E

+--+ +

E

+

++

+1

+

* Binding. inhibition of biotin-labelled MAbs by unlabelled antibodies as defined by + , > 7 5 ~ ; _+, 35 to 7 5 ~ ; - , - 3 5 to 3 4 ~ ; E, < -35%.

2068

D. H. Cybinski and others

other viruses w h i c h have been s h o w n to be serologically related to B E F V ( B R M , K I M a n d A R viruses) (Cybinski, 1987). T h e serological r e l a t i o n s h i p b e t w e e n B E F group viruses by n e u t r a l i z a t i o n tests using polyclonal a n t i b o d i e s is s h o w n in T a b l e 3. K I M a n d A R viruses, although k n o w n to be related by I F A ( G a r d et al., 1984) failed to cross-react in n e u t r a l i z a t i o n tests. Strong crossreactions were detected b e t w e e n B E F V a n d weaker cross-reactions were detected b e t w e e n B E F V strains a n d B R M virus. T w o ascitic fluids p r e p a r e d a g a i n s t B R M virus varied significantly in their p a t t e r n s of crossreactivity with B E F V strains. T h e results of neutralization a n d I F A tests on n e u t r a l i z i n g M A b s , using the same B E F group viruses, are shown in T a b l e 4. T h e results o b t a i n e d by n e u t r a l i z a t i o n tests o n ascites fluid generally agreed with those o b t a i n e d by I F A o n h y b r i d o m a s u p e r n a t a n t s with the exception of two a n t i b o d i e s reacting with epitopes in site G3 (8B6 a n d 8D3), which had low n e u t r a l i z i n g titres a n d low I g G c o n c e n t r a t i o n s . F o u r of the a n t i b o d i e s w h i c h recognize

distinct a n t i g e n i c sites on the B E F V G protein. All M A b s assigned to site G 1 reacted in i m m u n o b l o t s with a n t i g e n w h i c h had b e e n treated with SDS a n d 2-ME. M A b s assigned to site G2, however, reacted with G p r o t e i n only in the a b s e n c e o f 2-ME. T h e c o n f o r m a t i o n of site G3 also appears to be altered by t r e a t m e n t with 2ME. T h e four a n t i b o d i e s w h i c h b o u n d to site G3 were assigned to two subsets, G 3 a a n d G3b. A n t i b o d i e s w i t h i n each subset c o m p e t e d strongly, whereas some a n t i b o d i e s in different subsets showed partial competition. T h e b i n d i n g of b i o t i n y l a t e d 8B6 was i n h i b i t e d by seven heterologous c o m p e t i n g a n t i b o d i e s o n three a n t i g e n i c sites. However, six of these showed i n h i b i t i o n only at high a n t i b o d y c o n c e n t r a t i o n s ( > 10 ~tg/ml).

Serological reactions of MAbs with BEFV-related viruses Viruses analysed for cross-reactivity using polyclonal a n t i b o d i e s a n d M A b s were the BB7721 strain of B E F V , the insect isolate C S I R O 42 a n d C S I R O 53 a n d three

Table 3. Cross-neutralization titres of Australian rhabdoviruses related to BEFV

using polyelonal antibodies Virus Ascites fluid

BEF (BB7721)

BEF (CSIRO 42)

BEF (CSIRO 53)

BRM

KIM

AR

1024 64 512 192