Immunological relationships between rabies virus and rabies-related viruses studied with monoclonal antibodies to Mokola virus

Q ELSEVIER Paris 1988

Ann. Inst. Pasteur/Virol.

1988, 139, 157-173

IMMUNOLOGICAL RELATIONSHIPS BETWEEN RABIES VIRUS A N D RABIES-RELATED VIRUSES STUDIED WITH MONOCLONAL ANTIBODIES TO MOKOLA VIRUS J. Vincent, F. Bussereau (*) and P. Sureau Unitd de la Rage, Institut Pasteur, 75724 Paris Cedex 15

SUMMARY

Specific monoclonal antibodies (mAb) were prepared against a rabiesrelated lyssavirus, Mokola virus. A strain isolated in the Central African Republic, Mok-3, was used as immunogen. After 3 fusions more than 90 hybridoma cultures secreting mAb were identified. According to their different patterns of reactivity against rabies and rabies-related viruses, 61 ascites fluids were obtained. The antibody class was IgM for 2 of them and IgG for 59. They were specific for one of the 4 major viral proteins, as determined by immunofiuorescence, neutralization and immunoblotting tests. Their pattems of reactivity were determined against 6 different strains of rabies-related viruses: Lagos-bat virus from Nigeria (Lag-l) and the Central African Republic (Lag-2), Duvenhage virus from the Republic of South Africa (Duv-1) and Federal Republic of Germany (Duv-3), Mokola virus from Nigeria (Mok-1) and Cameroon (Mok-2) and a fixed strain of rabies virus, the challenge virus strain (CVS). According to their reactivities with these strains and the pattern of fluorescence, the mAb were classified into 11 different groups with intracytoplasmic fluorescence and 7 groups with cell surface fluorescence. A differential diagnosis of these lyssaviruses is possible in tissue culture using some of these mAb. KEY-WORDS; Lyssavirus, Mokola virus, Lagos-bat virus, Duvenhage virus, Rabies virus; Monoclonal antibodies, Diagnosis.

Submitted July 15, 1987, accepted December 2, 1987. (*) To whom reprint requests should be addressed.

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INTRODUCTION Three African viruses are related to the rabies virus. The first one discovered in 1956, the Lagos-bat virus (Lag), was isolated from bats in Nigeria [1] and then in the Central African Republic (CAR) [12, 13]. The second rabies-related virus, Mokola (Mok), was isolated from insectivores in Nigeria in 1968 [10], Cameroon [6] and then from a rodent in the CAR [8]. The third rabies-related virus, Duvenhage (Duv) was isolated in 1971 from a h u m a n case and from a bat in the Republic of South Africa [7] and then from European bats [9]. The relationship between the 4 viruses Lag, Mok, Duv and rabies (RV) was first determined on a morphological and serological basis using polyclonal antibodies. However, distinction between them can be made by quantitative experiments (for review see Wiktor and Hattwick [15]). Since (1) the standard diagnostic procedures, mouse inoculation or fluorescence obtained with a polyclonal fluorescent anti-rabies rabbit serum, do not distinguish rabies from rabies-related virus, (2) rabies and all rabies-related viruses except Lag are known h u m a n pathogens [3, 4, 7], and (3) conventional rabies vaccines protect against Duv virus but not against Mok [5], it was of primary interest to select a panel of anti-Mok monoclonal antibodies (mAb) that can be used for rapid differential diagnosis among all members of the lyssavirus group. This report describes the general properties of hybridomas produced against a Mok virus strain (Mok-3) isolated in the CAR. The isolate was obtained from the brain of a wild rodent Lophuromys sikapusi and identified as Mok virus by complement fixation and seroneutralization tests [8]. MATERIALS AND METHODS 1) Cells. Monkey cells, Vero type, and hamster cells, CH type, were grown in Dulbecco MEM supplemented with 2 % sodium bicarbonate, 1% antibiotics and antimycotic solution ( • 100) and 5 % foetal calf serum (FCS). BHK cells were grown in Eagle's MEM supplemented with 2 % sodium bicarbonate, 5 % FCS and 10 % tryptose phosphate broth.

BHK BSA CH CMM CSA CVS Duv FCS HAT i.c. IF i.p.

= = = = = = = = = = = =

b a b y h a m s t e r k i d n e y (cell). bovine serum albumin. h a m s t e r cell. complete mAb medium. cell s u r f a c e antigen. c h a l l e n g e virus s t r a i n (RV). D u v e n h a g e virus. foetal calf serum. hypoxanthine aminopterin thymidine. intracerebral(ly). immunofluorescence. intraperitoneal(ly).

Lag mAb MEM Mok NCA PAGE PBS PEG p.i. RV SDS

= = = = = = = = = = =

L a g o s - b a t virus. monoclonal antibody. minimal essential medium. Mokola virus. nucleocapsid antigen. p o l y a c r y l a m i d e gel electrophoresis. p h o s p h a t e - b u f f e r e d saline. p o l y e t h y l e n e glycol. post-infection. rabies virus. s o d i u m d o d e c y l sulphate.

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2) Viruses. a) Mouse passage. The different strains o f viruses were obtained from the National Reference Centre for Rabies (Institut Pasteur, Paris) as freeze-dried suckling mouse brain passage: Mok virus from Nigeria (Mok-1), Cameroon (Mok-2) and the CAR (Mok-3); Lag virus from the Nigeria (Lag-l) and CAR (Lag-2); Duv virus from the Republic of South Africa (Duv-l) and the Federal Republic of Germany (Duv-3); the challenge virus strain (CVS), a fixed strain of rabies virus. Three-day old suckling mice were inoculated intracerebrally (i.c.) with the different virus strains. Brains were harvested when complete paralysis occurred. A 10 070 brain suspension in Dulbecco medium (5 070 FCS) was clarified by centrifugation (10,000 g, 30 min). Virus containing supernatant was immediatly used for infection o f suckling mice or cells and stored at - 7 0 ~

b) Tissue culture. Cells to be infected with all the viruses except Lag were used as cell suspensions (Vero and BHK cells). A 2-ml sample of freshly trypsinized ceils (4 • 106 ml) was mixed with 6 ml of centrifuged brain suspension and incubated for 1 h at 37~ Cells were then centrifuged for 5 min at 250 g, the supernatant discarded and the cell pellet dispersed in 12 ml of growth medium. Two T25 flasks were seeded each with 5 ml o f the infected cell suspension. In parallel, 2 Terasaki 60-well plates were prepared (10 ~d/well) with the last 2 ml o f infected cell suspension. The viral growth was controlled in these Terasaki plates by immunofluorescence (IF) staining on acetone-fixed cells at 3 days post-infection (p.i.). If positive, the supernatant mediums of tissue culture flasks were harvested at days 5 and 10 p.i., dispensed in 2 ml aliquots and stored at - 70~ If necessary subsequent virus passages were made using the same protocol. C H cells were used for adaptation and passage o f Lag virus, which gave higher titres in these cells than in Vero or BHK cells. The cells were infected as confluent cell monolayers because they could not be easily obtained in homogeneous suspension after trypsinization (only large clumps were obtained). Cell monolayers in 2 T25 flasks were used. The virus, 1 ml o f mouse-brain-derived suspension, was left to adsorb for 1 h at 37~ Ceils were washed 3 times with medium and the flasks refilled with 5 ml of the same growth medium. At 5 and 10 days p.i., virus presence was checked, as for other viruses in Vero cells on Terasaki plates. When necessary, virus was produced in CH cells using the same protocol.

c) Virus titration. Ten-fold virus dilutions were prepared in 96-well plates (100 ~d/well, 4 wells/dilution). Then cells were added (4 • 104 cells in 100 ~d/well). After 3 days at 37~ infected cells were detected by IF. The virus infectivity titre was evaluated considering the highest virus dilution capable of infecting at least 10 ~ of cells.

d) Virus neutralization tests. Sera or ascites fluids were used. Tests were performed with variable quantities of antibody in the presence of a fLxed quantity of virus, producing 90 to 100 ~ fluorescent infected cells. Twofold dilutions starting at 1/10 of antibodies were made in 96-well plates (100 ~d/well, 2 wells/dilution). The viral suspension was added (100 ~d/well) and neutralized for 1 h at 37~ Then, cells were dispensed (4 • 104 ceils in 50 ~d/well). After 3 days at 37~ infected cells were detected by IF. The

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antibody neutralizing titre was evaluated considering the highest dilution of antibody capable of reducing the number of infected ceils by 50 ~ as compared to cells infected with the same quantity of non-neutralized virus.

e) Purification and labelling of Mok,3 virus. To avoid possible antigenic variations from the brain-passaged virus, virus stocks were produced with low tissue culture passages. Cells on the microcarriers were inoculated in suspension at a multiplicity of infection of 1 infective unit/ceU (as evaluated from fluorescent foci titration). After 1-h incubation at 37~ with intermittent manual shaking, the inoculated ceils were seeded (1-2• 107 ceUs/T150 flask). Between 2030 flasks were used for each production of purified virus. At days 5 and 10 p.i., medium was harvested and immediately centrifuged at 5,000 g for 30 min. The supernatant was recovered and ajusted to 5 070 sucrose final concentration with a sterile 50 070 sucrose solution. The virus was pelleted from the sucrose-supplemented viral suspension at 20,000 g for 14-16 h. The viral pellet was dispersed in a small volume of NT (sodium chloride 0.13 M, 0.05 M Tris-hydrochloride pH 7.8) and layered onto sucrose gradient (10-50 070 w/w in NT). After 1-h ultracentrifugation at 45,000 g, the virus band was collected and stored at - 7 0 ~ Virion morphology was verified by electron microscopy (fig. 1) and the degree of purification measured.

FIG. 1.

-

-

Mok-3 virus strain propagated in Vero cells, purified and prepared for negative contrast electron microscopy • 200, 000.

Micrograph courtesy of C. Dauguet,

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3) Production of hybridomas. a) Antigen. Three-day old suckling BALB/c mice were inoculated i.c. with Mok-3 virus. Brains were harvested in agonic phase and homogenized as a 10 % suspension in sodium bicarbonate-supplemented Dulbecco medium without serum and centrifuged at 10,000 g for 30 min. The supernatant was harvested and stored at 70~

b) Immunization procedure. Different immunization procedures using ~-propiolactone- or UV-inactivated viral brain suspensions were tested: 1)intrasplenic inoculation; 2)i.p. immunization without Freund adjuvant; 3) i.p. plus hind-foot pad inoculations without Freund adjuvant; 4)i.p. immunization with Freund adjuvant; 5)i.p. plus hind-foot pad inoculations with Freund adjuvant. The last procedure gave the best serum titres and was selected for use: a l-ml mixture in equal parts of UV-inactivated brain suspension and incomplete Freund adjuvant was inoculated i.p. into 2-month old BALB/c mice and 0.1 to 0.2 ml of the same mixture was also inoculated into the hind-foot pads of the mice. One month later the retro-orbital sinus was punctured and the level of blood antibodies was determined by indirect IF. Mice were then boostered i.p. with 1 ml of (UV-inactivated or not) brain suspension and boosters were given as often as necessary to reach an antibody blood level of 1/2,500-1/5,000. Blood antibody level was checked every 3 weeks, until it dropped to approximately 1/5-1/10 of the maximum titre. Then, the last i.p. booster before fusion was given (0.5 ml) followed 1 h later by intravenous injections (0.2 ml) of not-inactivated viral brain suspension. Spleen was removed 3 days later for fusion. c) Culture medium for hybridization. Sp2/0-Agl4 or P3-X63-Ag8.653 (X63-Ag8) myeloma cells and hybridomas were grown in complete mAb medium (CMM): Dulbecco medium supplemented with 10 ~ FCS, glutamin (2 07o 200 mM solution), vitamins (1 ~ • 100) 1 ~ sodium pyruvate and 2 ~ sodium bicarbonate. In selective isolation of hybridomas, CMM was used diluted in equal parts with 24-h myeloma-cell-conditionedmedium and supplemented with HAT medium (2 ~ x 50). d) Fusion. Briefly, fusion was carried out according to the protocol described in ~ Hybridoma Techniques~ (EMBO, SKMB Course 1980 Basel), but at a ratio of 1/1 splenocytes and myeloma cells with 1 ml 50 ~ polyethylene glycol (PEG) 1500.

e) Clones. After fusion and elimination of PEG, the cells were suspended in 50 ml of selective medium and 0.1 ml of the suspension dispensed in each well of five 96-well plates seeded the day before with 1 x 104 adult BALB/c mice peritoneal macrophages/well. Half the medium of each well was changed every 5 days. On days 15 or 30 after fusion, the supernatants of all the wells where hybridoma cell growth took place were screened by IF antibody staining for the presence of specific antibodies to Mok-3 on Mok-3-infected Vero cells in Terasaki plates.

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Antibody-producing hybridomas were amplified first on mouse peritoneal macrophage feeder layers, then without macrophages. Hybridomas growing easily in absence of macrophages and still producing specific antibodies (established hybridomas) were frozen in liquid nitrogen.

f) Subclones. With all established hybridomas, subcloning was performed by limiting dilutions in CMM in 96-well plates seeded the day before with I x 104 BALB/c mice peritoneal macrophages/well ; all the wells with growing cultures were checked 11-12 days after subcloning for antibody production. The monoclonal nature of subclones was assessed by considering the statistical distribution of positive wells in the original microplate and in the subclones. Second subcloning was performed when the probability of a Poisson distribution with mean > 1 could be evoqued from the distribution of positive wells in subclones.

4) Isotype determination. Isotypes were determined by double immunodiffusion tests with hybridoma supernatants : 1 volume of each supernatant was added to 1 volume of ammonium sulphate pH 7.6. The mixture was allowed to react for 3 h at 4~ and was then centrifuged at 10,000 g for 20 rain. The pellet was resuspended in distilled water (1/10 initial volume) and the concentrated antibody preparation was reacted in 2 % Indubiose A37 plaques against isotype-specific antisera (Miles Scientific).

5) Production of ascites. Working preparations of antibody were obtained by production of ascites fluids in 10-week old BALB/c mice pretreated with pristane (2, 6, 10, 14 tetramethylpentadecane) 15 days before i.p. inoculation of hybridomas. After collection, ascites fluids were stored overnight at 4~ and cells were eliminated by centrifugation (5,000 g, 30 rain). Ascites fluids corresponding to same subclone and day of mouse inoculation were pooled and stored in aliquots at - 2 0 ~

6) mAb titration. Serial 2-fold dilutions of ascites (from 1/80 to 1/40,000) were distributed in duplicate on Mok-3-infected Vero cells (Terasaki plates) and antibody titres were determined by the fluorescent focus reduction test [14].

7) Virus neutralization. Serial 2-fold dilutions of pre-diluted heat-inactivated ascites fluids (from 1/80 to 1/40,000) were distributed in 96-weU plates (100 rd/well) and mixed with an equal volume of a virus dilution necessary to obtain < 100 % infected cells. The plates were incubated for 1 h at 37~ Trypsinized Veto cells were added (2 x 103/well). After 3 days p.i., cells were acetone-fixed and neutralization was determined by the fluorescent focus reduction test [14].

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8) Immunofluorescence tests. After 3 washes with phosphate-buffered saline (PBS) containing Ca + + and Mg + + ( + Ca + § + Mg + +), infected cells were fixed in situ (Terasaki, microplate or slides) with 80 % acetone (4~ in distilled water at r o o m temperature for 30 min, then the fixative was poured o f f and the plate dried at r o o m temperature. Acetonefixed preparations were stored at - 2 0 ~ To test hybridoma supernatants, the frozen plates were thawed and washed twice in 0.05 % Tween-20/PBS ( + Ca* + + Mg + +) prior to the addition of culture supernatant. After a 30-rain incubation at 37~ with the supernatant, the plates were washed 3 times with PBS ( + Ca 9 + + Mg + +) before addition of the conjugate. To test ascites fluid dilutions, the frozen plates were thawed and washed as previously described, then treated with 0.1 070 B S A / P B S ( + C a ++ + M g ++) for 30 rain. Then, the plates were washed with PBS p H 7.5. After a 30-min incubation at 37~ with ascites fluid, the plates were washed as for supernatants except that PBS without Ca + + + Mg+ + was used. Then, conjugate was added. Direct and indirect fluorescent antibody staining were performed: in the first case with fluoresceinisothiocyanate-labelled rabbit anti-rabies polyclonal globulins (Diagnostics Pasteur) ; in the second case with anti-mouse purified IgG fluorescein isothiocyanate conjugate (Diagnostics Pasteur). All incubation times were 30 min. Washed and dried plates were examined using an epifluorescence microscope. Localization of m e m b r a n e components was studied after fixation using paraformaldehyde in solution (3 % in PBS + Ca + + + Mg + +, p H 7.4) or on non-fixed cells. The protocol for paraformaldehyd e fixation was as follows. Cells were washed twice with PBS then fixed at r o o m temperature for 20 min. After treatment cells were washed twice with PBS, once with 50 m M NH4C1/PBS and twice again with PBS. At this point paraformaldehyde-fixed cells and non-fixed cells were tested with ascites fluids and stained by indirect IF, as previously described.

9) Western blotting. Viral proteins were separated by S D S - P A G E (12 o70 polyacrylamide gel, 16-17 h at 25 m A / g e l with permanent refrigeration). After electrotransfer to a BA 85 nitrocellulose sheet (Schleicher-Schull), non-specific binding sites were saturated with 5 % skimmed milk in PBS, 3 m m wide strips were cut in the nitrocellulose sheet, incubated for 1 h at r o o m temperature and subsequently overnight at 4~ with permanent 3-dimensional rocking, with ascite fluid diluted 1/100 in 5 % skimmed milk in PBS. After 3 washes with PBS containing 0.05 % Tween-20, the nitrocellulose strips were treated for 1 h at r o o m temperature with anti-mouse IgG peroxidase conjugate (Diagnostics Pasteur) diluted according to the m a n u f a c t u r e r ' s instructions. After 3 washes with Tween-20/PBS, diamino benzidine was added as a substrate and the reaction was stopped after 10-20 min by washing the strips with distilled and tap water.

RESULTS AND DISCUSSION Production of hybridoma cultures. M o k a n d D u v viruses w e r e easily a d a p t e d to V e r o cells with m a x i m a l infectious titres o f 1 • 106/ml. L a g virus grew very p o o r l y in V e r o cells b u t gave a p p r o x i m a t e l y x 100 t h e titre o b t a i n e d in V e r o cells in C H cells.

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Considering the rather low titres obtained for all strains in tissue culture, we preferred the brain-derived antigen obtained after Mok-3 i.c. inoculation of BALB/c suckling mice for the immunization procedure, instead of purified Mok-3 virions from cell supernatants. As the UV-irradiated antigen when used in the first injection of mice produced better serum titres than [3-propiolactonetreated antigens, UV-irradiated antigen was used for i.p. plus hind-foot pad inoculation with Freund adjuvant. Fusion 1 was obtained after boosters with inactivated antigen, and fusions 2 and 3 after boosters with non-inactivated antigen; boosters with non-UV-inactivated antigens induced higher blood titres than boosters with inactivated antigen. The highest percentage of hybrid growth was obtained after fusions with X63-Ag8 myeloma cells (fusions 2 and 3) compared to fusion with SP2/0-Agl4 (fusion 1). The greatest percentage of stable mAb-producing clones after 10-15 subcultures was obtained in fusions 2 and 3 ; 12 clones out of 21 detected at day 15 after fusion in fusion 2, and 60 out of 90 in fusion 3. Each established clone was subcloned, and 3-5 mAb-producing subclones were amplified in tissue culture and frozen in liquid nitrogen.

Isotype determination. The isotype of each subclone was determined using hybrid cell supernatant. Fusion 1 yielded mainly IgM. For fusions 2 and 3 only IgG were found to be represented, mAb of IgG1 being the most common. Listed in table I are the antibody classes corresponding to the subclones for which the ascites fluids were selected for rabies and rabies-related virus identification. Fifty mAb belonged to the IgG1 subclass, 5 to subclass IgG2a, 3 to subclass Ig2b, 1 to subclass IgG3 and 2 to IgM. All the mAb produced by subclones deriving from the same original clone corresponded to the same antibody class or subclass with 2 exceptions: 1) subclone 4-4 belonged to the immunoglobulin subclass IgG2a, whereas subclones 4-5, 4-6 and 4-7 appeared to be of the IgG3 class ; 2) subclones 80-1 and 80-4 were IgG1 and subclones 80-7, 80-15 and 80-17 IgG2a (data not shown).

FIG. 2. -- Localization of viral antigen in Vero cells at 3 days p.i. Intracytoplasmic fluorescencewas observed on acetone-fixedcells infected with: A) Mok-1 (mAb 22-3); B) Duv-I (mAb 31-6); C) Mok-3 (mAb 31-6). Membrane fluorescencewas observed either on acetone-fixedceils infected with: D) Mok-3 (mAb M24); E) Mok-2 (mAb M24); or in paraformaldehyde-treatedcells infected with (F) Mok-3 (mAb 53-21).

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lmmunospecificity o f hybridoma supernatant. The ability of the mAb to detect viral antigens at different localizations of Mok-3-infected cells was tested with an indirect IF test. We defined 3 different patterns of fluorescence : 1) a fine granular, cytoplasmic staining clearly visible as soon as 24 h p.i., involving the whole cytoplasm with very often small inclusion bodies (fig. 2B and 2C); 2) a coarse granular cytoplasmic staining (fig. 2A) ; 3) a very diffuse staining underlining the periphery of some of the cells, faintly visible 1-2 days p.i. but more extensive and clearly visible 3-5 days p.i. (fig. 2D and 2E). In this case, very fine granular cytoplasmic staining was also present. Considering the results obtained with other rhabdoviruses such as vesicular stomatitis virus or RV, the first 2 fluorescence patterns were assigned to an equivalent of nucleocapsid antigen (NCA) and the third one to G viral antigen expressed at the surface of the cell membrane (CSA). In all the mAb screening tests, monolayers were fixed at 2 and 5 days p.i. to discriminate clearly the fluorescence pattern. All the mAb produced by subclones derived from the same original clone had the same corresponding fluorescence pattern, with 2 exceptions: 62-1 NCA pattern and 62-47 CSA pattern. Production of ascites fluids. Fifty-one different clones were selected according to the specificities of the hybridoma cell supernatants using cells infected with 2 rabies-related viruses Lag and Duv, and a fixed strain of RV. For each clone, subclones giving the highest tissue culture supernatant titres against Mok-3 were amplified and transferred to pristane pretreated BALB/c mice. In some instances when low titres in the first ascite fluid was obtained, multiple subclones originating from the same clone were transferred to mice. Ascites fluids were titrated by end-point fluorescence titration on Mok-3-infected Vero cells. Titres varied between 1/640-1/80,000 (table I). The lower

TABLEJ. Designation (i) M2 M7 M15 M17 M18 M24 M27 M28

-

-

Properties of anti-Mok-3 virus mAb.

Isotype (Ig)

Fluorescence

Blotting ~2)

Titre (3)

M G2a M G2b G1 G2a G1 G1

NCA NCA NCA CSA NCA CSA NCA NCA

ND ND M1 Ml

1/ 1,280 1/640 1/640 1/10,000 > 1/40,000 > 1/40,000 1/40,000 1/40,000

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D e s i g n a t i o n (1)

Isotype (Ig)

Fluorescence

B l o t t i n g (2)

Titre (3)

M30 4-4 4-7 (*) 7-4 (**) 9-4 11-1 17-3 18-4 22-3 23-10 24-3 26-9 28-4 (**) 29-1 31-6 43-7 (**) 44-1 51-2 (**) 53-21 59-1 62-1 62-47 (*) 68-10 69-9 71-5 76-6 (**) 80-4 91-6 98-1 105 -20 111-14 116-10 121-6 128-19 131-8 135-18 (**) 153-8 (**) 181-6 183-14 184-14 185-10 186-24 (**) 189-12 190-3 244-11

G1 G2a G3 G1 G1 G1 G1 G1 G2a G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G1 G2b G1 G1 G1 G1 G1 G1 G1 G1 G1 G2a

NCA NCA NCA CSA NCA NCA NCA NCA NCA NCA NCA NCA NCA CSA NCA NCA NCA NCA CSA NCA NCA CSA NCA NCA NCA NCA NCA CSA NCA NCA NCA CSA NCA NCA NCA CSA NCA NCA NCA NCA NCA CSA CSA NCA CSA

M1 M1 M1 N M1 M1 M1 M1 M1 M2 M1 ND M2 M1 N M2 M2 M1 N M2 M1 M1 M1 M1 M2 M2 ND -

1/5,000 1/2,560 1/640 1/5,120 1/5,120 1/5,120 1/40,000 1/2,560 1/2,560 1/2,560 1/5,120 1/640 1/5,120 1/5,120 1/10,240 1/5,120 1/2,560 1/2,560 1/2,560 1/2,560 1/2,560 1/5,120 1/640 > 1/80,000 1/5,120 1/1,280 1/5,120 1/!,280 1/5,120 1/5,120 1/20,500 1/5,120 1/205 1/1,280 1/2,560 1/1,280 1/2,560 1/10,000 1/640 1/20,500 1/640 1/10,000 1/320 1/2,560 1/5,120

(1) Fusion 1 corresponds to subclones M2, M7 and M15. Fusion 2 corresponds to M17-M30. For fusion 3 (2- or 3-number designations), the first number refers to the original clone and the following one(s) to the subclone choosen, (2) ND = not done; - = no blotting obtained; N = nucleocapsidprotein; M1 and M2 = membrane proteins. (3) Ascites fluids titre determined by fluorescent focus reduction tests. (*) Subclones from the same clone with different characteristics. (**) Subclones from the same clone with the same characteristics: same titre (135-10 and 153-12); different titre 7-10 (1/640), 28-5 (1/2,560), 43-8 (1/!,280), 51-9 (1/1,280), 76-6-4(1/40,000), 186-20(1/2,560).

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titres were probably related to clone instability which appeared late in the subcultures with the number of mAb-producing cells being progressively reduced. For example, subclone 76-6 was submitted to an extra subcloning : only 10 % of the sub-subclones were mAb producers but the ascite fluid obtained with one of them, 76-6-4, yielded a high titre (1/40,000) compared with that obtained with subclone 76-6 (1/1,280). All the subclones that gave ascites fluids with low titres are now undergoing additional subclonings. Sixty one ascites fluids were selected (lst fusion, 3 ; 2nd fusion, 6; 3rd fusion, 52). A m o n g them, 51 ascites fluids corresponded to different original clones, and 10 to duplicate subclones.

Fluorescence characteristics of ascites fluids.

As expected, the patterns of fluorescence obtained with ascites fluids corresponded to those of hybridoma culture supernatants (table I). To confirm the position of the m A b targets displaying CSA fluorescence on acetone-fixed ceils, 2 types of infected cells were used : non-fixed cells and paraformaldehyde-fixed cells at 3 days p.i. Dilutions of ascites fluids were also assayed on the Mok-3-infected ceils. For all CSA-type ascites fluids on non-fixed ceils, the membrane fluorescence aspect was fine granular and distributed over some parts of the surface of the cell. No fluorescence was obtained with m A b giving NCA-type fluorescence. On paraformaldehydefixed cells, granular cell surface fluorescence was also visible with m A b displaying CSA fluorescence, and was more intense than that obtained on unfixed cells, probably involving repetitive epitopes more deeply situated inside the cell membrane which became accessible to m A b after paraformaldehyde fixation (fig. 2F). Intracytoplasmic fluorescence was also visible with NCAtype mAb, showing that after such a fixation the membrane of Mok-3-virusinfected cells became permeable to antibodies. The cellular position of the target antigens of different m A b were not determined conclusively after paraformaldehyde fixation, and only fluorescence on acetone-fixed or nonfixed cells differentiated CSA and NCA patterns of fluorescence.

Neutralization test.

The ability of the 15 mAb giving a CSA pattem of fluorescence to neutralize the 8 different strains was assayed. Only one mAb, M-24, had neutralizing capacity. The 3 Mok strains were completely neutralized at 1/40,000 dilution. By IF test, M-24 recognized Mok-3 and Mok-1 but not Mok-2. This might be due to a difference in expression of the neutralizing epitope on the cell membrane and on the envelope. This lack of correlation was not due to the fact that different virus passages in Vero cells were used, as same results (in IF or neutralization tests) were obtained whatever the virus adaptation

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to cell cultures (1st up to the 5th passage). The fact that only one mAb had neutralizing activity may be due to the immunization material as brain derived antigen was used instead of virions.

Polypeptide specificity. The purified preparation of Mok-3 virions was resolved into 4 protein species : the G glycoprotein, the N nucleoprotein, the M1 and M2 membrane proteins [11]. The reactive proteins were further assayed in Western blotting. Immunoblotting was obtained at the respective positions of the N, M1 and M2 proteins, but not at the position of the G protein (table I). This lack of reactivity was not related to possible low titre of ascites fluids because ascites fluids 26-9 (1/640), 68-10 (1/640) and 189-12 (1/320) produced a strong reaction whereas 69-9 produced a negative one ( > 1/80000). No blotting was obtained in particular with M-24 directed against a neutralizing epitope of G protein. This epitope must be conformational as well as those corresponding to M7, M17, M18, 7-4, 9-4, 18-4, 24-3, 44-1, 51-2, 51-9, 53-21, 59-1, 62-1, 62-47, 69-9, 80-4, 98-1, 116-10, 135-18, 184-14, 185-10 and 244-11. Eight CSA-type fluorescence m A b out of 12, recognized conformational epitopes. The others (29-1, 91-6, 186-20 and 24, 189-12) were directed against the M2 protein; this protein, in the case of RV, has been shown to be located at the cell membrane [2]. Twelve NCA-type fluorescence m A b out of 40 recognized conformational epitopes. The others were directed against the N, M l or M2 proteins.

Reactivity of ascites fluids with iyssaviruses. To determine their patterns of reactivity to the different viruses, standard dilution was determined for each ascites fluid. Standard dilution corresponded to an antibody concentration at least 4 times below the titration end-point on Mok-3 infected cells. Then, the m A b were screened on 2-fold dilutions below this point by IF tests. Positive reaction was noted when the 4 dilutions gave bright fluorescence. The m A b reacted to the same titre with homologous and heterologous viruses. Negative results were then controlled with ascites fluids diluted so as to give an antibody concentration at least 16-64 times below the standard dilution; the control was also extended to 1/40 or 1/80 dilutions. Lack of reactivity to normal cells components was also checked with ascites fluids diluted only 1/20 to 1/40 on confluent non-infected Vero and BHK cells. None of the 61 m A b reacted with cellular components. The ascites fluids were screened at different dilutions in IF tests for immunological reactivity against 7 strains : Mok (strains 1 and 2), Lag (strains 1 and 2), Duv (strains 1 and 3) and CVS. CVS was choosen as this RV strain is generally used for vaccine potency tests.

J. V I N C E N T A N D COLL.

170

m A b giving a N C A fluorescence pattern were categorized into 11 groups (table II). G r o u p 1 consisted o f m A b which cross-reacted only with Mok-3 strain. G r o u p 9 permitted us to distinguish between rabies-related viruses and RV. Group 12 consisted of m A b which cross-reacted with all 8 strains. Association of representative m A b of different groups allowed identification o f every strain used in this experiment : groups 1, 2 and 4 identification o f the M o k strains; groups 3 (or 6), 4 (or 5), 7 differentiation o f the Lag strains ; groups 5 (or 10), 8 (or 11) differentiation o f the Duv strains ; groups 9 (or 10, 11) identification o f CVS strain.

TABLE II. - - Reactivity by IF test o f M o k - 3 mAb (NCA pattern) with different lyssavirus strains.

Group

Rabies CVS

3

Mokola 2 1

Lagos-bat 2 1

Duvenhage 3 1

Nb of different clones

1

0

+

0

0

0

0

0

0

7

2 3

0 0

+ +

+ 0

0 0

0 +

0 0

0 0

0 0

2 1

4

0

+

+

+

0

0

0

0

4

5 6 7

0 0 0

+ + +

+ + +

+ + +

0 + +

0 0 +

+ 0 0

0 0 0

1 3 4

8

0

+

+

+

+

+

+

+

1

9 10 11

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

0 + +

0 0 +

5 5 8

+ corresponds to a positive reaction in cells infected with each virus with an ascite fluid to ascites used at a concentration 2-4 times below the end point. 0 corresponds to a negative reaction to ascitic fluid used at a concentration 16-64 times below the end point. mAb included in each group: 1) M18, 4-4, 18-4, 24-3, 68-10, 80-4, 153-8, 153-12; 2) M-15, 69-9; 3) 4-7; 4) 17-3, 43-7, 43-8, 131-8, 183-14; 5) 111-14; 6) M2, M7, 71-5; 7) 51-2, 51-9, 62-1, 98-1, 190-3; 8) 26-9; 9) ll-1, 44-1, 76-6, 76-6-4, 121-6, 184-14; 10) M27, M28, M30, 23-10, 28-4, 28-5; 11) 9-4, 22-3, 31-6, 59-1, 105-20, 128-19, 181-6, 185-10.

m A b giving C S A fluorescence pattern were categorized into 7 groups (table III). Strain identification was possible for M o k , Lag, Duv and CVS. Using the IF test, Mok-3 m A b o f b o t h types o f pattern reacted equally with the 8 different strains : N C A groups 1, 2, 4, 6, 7 and 11 and CSA groups 1, 2, 3, 4, 5 and 11, while the others were specific to the N C A groups 3, 5, 8, 9, 10 and CSA group 6. m A b produced by subclones derived from the same clone (8 duplicate clones: 7, 28, 43, 51, 76, 135,153, 186) were included in the same identifica-

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171

TABLE III. - - Reactivity by IF test o f Mok-3 m A b (CSA pattern) with different lyssavirus strains.

Rabies CVS

3

1

0

+

0

0

0

0

0

0

1

2 3 4 5 6 7

0 0 0 0 0 +

+ + + + + +

+ + + + + +

0 + + + + +

0 0 + + + +

0 0 0 + 0 +

0 0 0 0 + +

0 0 0 0 0 +

1 3 I 5 1 2

Group

Mokola 2 1

Lagos-bat 2 1

Duvenhage 3 1

Nb of different clones

+ corresponds to a positive reaction and 0 to a negative reaction. mAb included in each group: 1) 244-11 ; 2) M24; 3) M17; 4) 116-10; 5) 7-4, 7-10, 53-21, 62-47, 91-6, 189-12; 6) 135-10, 135-18; 7) 29-1, 186-20, 186-24.

tion group. They also had the same isotype polypeptide specificity and fluorescence pattern, m A b produced b y subclones derived from 2 clones reacted differently: 4-4 and 4-7 which had different isotypes respectively in groups 1 and 3 ( N C A - t y p e fluorescence) but were specific to the M1 protein; 62-1 and 62-47 displaying different types of fluorescence, respectively N C A (group 7) and CSA (group 5), reacted with the same virus strains, had the same isotype but polypeptide specificities could not be determined. Because of their high reactivity and specificity we expect that these antibodies will be very useful in tests for rapid diagnosis o f rabies-related viruses on brain tissue impressions. Determination o f a complete pattern o f m A b reactivities to Mok-3 in tissue culture or on brain smears towards all the rabies fixed strains available in the laboratory, street rabies virus from different geographical origins and additional rabies-related strains, is in progress.

RI~SUMI~ RELATIONS IMMUNOLOGIQUES ENTRE LE VIRUS RABIQUE ET LES VIRUS APPARENTt~S, A L'AIDE D'ANTICORPS MONOCLONAUX ANTI-MOKOLA

Des anticorps monoclonaux ont 6t6 obtenus avec le virus Mokola, un lyssavirus apparent4 au virus rabique. U n e souche isol6e en R6publique Centrafricaine (Mok-3) a 6t6 utilis6e c o m m e antighne. A la suite de 3 fusions, plus de 90 hybridomes secr6tant des anticorps m o n o c l o n a u x ont 6t6 caract6ris6s. A u regard de la r6activit4 avec le virus rabique et les virus apparent6s, 61 liquides d'ascites ont &6 produits. Deux appartiennent h la classe des IgM et 59 ~ celle des IgG. P a r immunofluorescence, neutralisation et <
172

J. V I N C E N T A N D COLL.

blot)>, il a 6t6 montr6 que chaque anticorps monoclonal est sp6cifique d'une des 4 prot6ines majeures du virion. Le profil de r6activit6 de chaque anticorps a 6t6 d6termin6 vis-a-vis de 6 souches de virus apparent6s : le virus Lagosbat du Nig6ria (Lag-l) et celui de R6publique Centrafricaine (Lag-2), le virus Duvenhage d'Afrique du Sud (Duv-1) et celui de R6publique F6d6rale d'Allemagne (Duv-3), le virus Mokola du Nig6ria (Mok-1) et celui du Cameroun (Mok-2) et une souche fixe de virus rabique, CVS. En tenant compte de leur r6activit6/~ l'6gard de ces souches ainsi que du type de fluorescence, les anticorps monoclonaux ont 6t6 class6s en 11 groupes diff6rents ayant une fluorescence intracytoplasmique, et en 7 groupes diff6rents ayant une fluorescence membranaire. Avec ces anticorps monoclonaux un diagnostic diff6rentiel de ces lyssavirus est possible en culture de cellules. MOTS-CLI~S: Lyssavirus, Virus Mokola, Virus Lagos-bat, Virus Duvenhage, Virus rabique; Anticorps monoclonaux, Diagnostic.

ACKNOWLEDGEMENTS The authors are grateful to the following people for kindly providing them with material: J.C. Mazie (SP2/0-Agl4 and P3-X63-Ag8.653), J.C. Guillon (CH cell line), G. Chevallier (Mok-3 virus production for purification), C. Dauguet (electron microscopy), S. Michelson (UV microscope), P. Picouet (photography); and to P. Gregorian for typing the manuscript.

REFERENCES

[1] BOULGER,L.R. & PORTERFIELD,J.S., Isolation of a virus from Nigerian fruit bats. Trans. roy. Soc. trop. Med. Hyg., 1958, 52, 421-424. [2] DELAGNEAU,J.F., PERRIN, P. • ATANASIU,P., Structure of the rabies virus; spatial relationshilSs on the proteins G, M1, M2 and N. Ann. Virol. (Inst. Pasteur), 1981, 132 E, 473-493. [3] FAMILUSI,J.B. & MOORE, D.L., Isolation of a rabies-related virus from the cerebrospinal fluid of a child with aseptic meningitidis. Afr. J. Med. Sci., 1972, 3, 93-96. [4] FAMILUSI,J.B., OSUNKOYA,B.O., MOORE, D.L., KEMP, G.E. & FABIYI, A., A fatal human infection with Mokola virus. Amer. J. trop. Med. Hyg., 1972, 21, 959-963. [5] LAFON,M., HERZOG,M. & SUREAU,P., Human rabies vaccines induce neutralizing antibodies against the European bat rabies virus (Duvenhage). Lancet, 1986, II, 515. [6] LE GONIDEC, G., RIC~ENBACH, A., ROBIN, Y. & HEME, G., Isolement d'une souche de virus Mokola au Cameroun. Ann. Microbiol. (Inst. Pasteur), 1978, 129 A, 245-249. [7] MEREDITH,C.D., ROSSOUW,A.P. & VAN PRAAGKOCH, H., An unusual case of human rabies thought to be of Chiropteran origin. S. Afr. med. J., 45, 767-769. [8] SALtJZZO,J.F., ROLLIN, P.E., DAUGUET,C., DIGOUTTE,J.P., GEORGES,A.J. & SUREAU, P., Premier isolement du virus Mokola /l partir d'un rongeur (Lophuromys sikapusi). Ann. Virol. (Inst. Pasteur), 1984, 135 E, 57-66.

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[9] SCHNEIDER,L.G., A new case of bat rabies in Germany. Rabies Bull. Europe., 1982, 4, 17-18. [10] SHOPE, R.E., MURPHY, F.A., HARRISON, A.K., CAUSEY, O.R., KEMP, G.E., SIMPSON, D.I. & MORE, D.L., Two African viruses serologically and morphologically related to rabies virus. J. Virol., 1970, 6, 690-692. [11] SOKOL, F. & KOPROWSKI, H., Structure-function relationships and mode of replication of animal rabdoviruses. Proc. nat. Acad. Sci. (Wash.), 1975, 72, 933-936.

[12] SUREAU,P., GERMAIN,M., HERVE, J.P., GEOFFROY,B., CORNET,J.P., HEME, G. & ROBIN, Y., Isolement du virus Lagos-bat en Empire Centrafricain. Bull. Soc. Path. exot., 1977, 70, 467-470. [13] SUREAU, P., TIGNOR, G.H. & SMITH, A.L., Antigenic characterization of the Bangui strain (ANCB-672d) of Lagos-bat virus. Ann. Virol. (Inst. Pasteur), 1980, 131 E, 25-32. [14] SUREAU,P., ROLLIN,P.E. & ZELLER, H., Corr61ations entre l'6preuve immunoenzymatique, la s6roneutralisafion et la r6duction de foyers fluorescents pour le titrage des anticorps rabiques. Comp. Immun. Microbiol. Infect. Dis., 1982, 5, 143-150. [15] WIKTOR,T.J. & HATTWlCK,M.A.W., Rhabdoviruses: rabies and rabies-related viruses, in ~