Protective activity of a murine monoclonal antibody against European bat lyssavirus 1 (EBL1) infection in mice

Protective activity of a murine monoclonal antibody against European bat lyssavirus 1 (EBL1) infection in mice J u a n A n t o n i o M o n t a f i o - H i r o s e , Mireille L a f a g e , P a t r i c k W e b e r , H a s s a n Badrane, Noel Tordo and Monique Lafon*

A mouse model was designed to test in vivo the efficacy of rabies immune globulins and specific neutralizing monoclonal antibodies to prevent European bat lyssavirus I infection. Human or equine rabies immune globulins previously found to contain variable amounts of neutralizing bat lyssavirus crossreactive antibodies were passively transferred to mice receiving intramuscularly a lethal dose of bat lyssavirus type 1. Immune globulins did not protect mice well against bat lyssavirus 1 whereas they reduced the mortality caused by rabies virus. In contrast, mice inoculated with bat lyssavirus 1 or rabies virus survived when passively immunized with bat lyssavirus 1 specific monoclonal antibody (mAb 8-2). This monoclonal antibody, an IgG2a, recognized an epitope located in the antigenic site IIa of rabies glycoprotein. A mutation replacing the lysine 198 by glutamate in a rabies variant abrogated sensitivity to this neutralizing antibody. Because of its broad neutralizing spectrum against wild virus isolates, including European bat lyssaviruses, this monoclonal antibody should be a good candidate for rabies immune globulin replacement. It could improve efficacy of rabies vaccination, used either alone or in conjunction with human rabies immune globulins or monoclonal antibody cocktail to supplement their lack of crossreactivity to European bat lyssavirus 1. Keywords:European bat lyssavirus;monoclonalantibodies;passive immunization;rabies virus; neutralization

Effective postexposure prophylaxis of rabies includes the local application of rabies immune globulins (RIGs) together with the first dose of vaccine. Efficacy of RIGs as a complement to vaccination has been demonstrated in animals 1'2 and after retrospective analysis of human treatments 3. RIGs are believed to neutralize extracellular infectious virus and to participate in the destruction of infected cells by antibody-dependent cellular cytotoxicity (ADCC). RIGs can be of either equine (ERIGs) or h u m a n (HRIGs) origin. Despite purification,potential risks are associated with their use: anaphylactic reactions related to ERIGs are still reported4 and the possibility of transmission of human pathogens such as hepatitis, Creutzfcld-Jakob disease or human immunodcficiency viruses as a consequence of inappropriate inactivation procedures or inadequate choice of donors is a major concern. Replacement of H R I G s by monoclonal antibodies (mAbs) of h u m a n or murine origin has been proposed s-8. Murine m A b s arc well tolcrated9 and have Unit6 de la Rage, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France. *To whom correspondence should be addressed. (Received 5 December 1992; revised 16 February 1993; accepted 1 March 1993)

several advantages over RIGs: they are easily standardized, and efficient at a lower protein concentration; moreover, they can be selected according to their neutralizing capacity in order to adapt the treatment to the nature of the virus, especially when infectious virus is antigenically different from the vaccine strains. Since 1968, two new types of lyssaviruses, the European bat lyssaviruses 1 and 2 (EBL1 and EBL2) 1°'tl have been isolated from bats in Europe. These viruses have been responsible for human deaths 12'13. They are clearly different at the antigenic ~4 and molecular ~s level from the vaccine strains. In the absence of EBL-specific vaccine, concern arose about prophylaxis of exposure to rabid European bats. Potency tests in mice indicated that, in contrast to animals vaccinated with PV or ERA vaccines, those receiving PM or LEP vaccines were not protected against EBL1 challenge 16-~s. In humans, although rabies vaccination induces EBLl-specific antibodies in some individuals 19-zl, they could not be detected in sera of 27% of rabies-immunized patients; moreover, their peripheral blood lymphocytes could not be stimulated in vitro with EBL1 antigen 22. Initially, HRIGs were thought to be able to supplement the lack of rabies vaccination crossreactivity. However, the HRIG contents of EBLl-specific antibodies were very variable

0264-410X/93/12/1259-08 © 1993 Butterworth-Heinemann Ltd

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Rabies serotherapy in mice: J.A. Monta~o-Hirose et al.

and sometimes almost negligible 22, suggesting that another source of EBL-specific immune globulins is needed. In this paper, the authors studied the possibility that murine mAbs directed against the envelope glycoprotein (G) and exhibiting a strong neutralizing activity against EBL1 could be used for EBL prophylaxis in complement to mAbs cocktails or to the present RIGs. MATERIALS AND METHODS

Virus strains Laboratory and wild strains of rabies virus were propagated in BSR cells 23, grown in minimal Eagle's medium (MEM) supplemented with L-glutamine, 8% fetal calf serum and gentamicin. Wild strains of rabies virus were isolated from foxes, badgers, sheep and horses in Yugoslavia in 1986 and 198824'2s and from foxes and raccoon dogs in the north of Poland in 1986 (Olsztyn area). Bat lyssaviruses were isolated from Eptesicus serotinus or unidentified bat species in several European countries: (Denmark [DAN bat], the former West [Stade isolate] and East Germany [DDR bat], Spain [SPA bat], France [FRA bat], Poland [POL bat 85]) and from Myotis dasycneme (Netherlands bats R30 and R8) (for references see Ref. 25). POL bat 90 is a new bat strain isolated in Poland in 1990. For serotherapy experiments, a German bat (Stade isolate 16) was used as a prototype of EBL1 and CVS obtained from the American Type Cell Collection (ref vr322) and PV strain ~s as prototypes of vaccine rabies virus strains. Monoelonal antibodies Hybridomas secreting EBLl-specific mAbs were generated by fusion of non-secreter murine myeloma SP20 Ag cells with splenocytes of Balb/c mice immunized with EBL1 according to procedures previously described zr. IgG were purified from ascites fluid by precipitation using the salting-out method with 42% saturated ammonium sulfate. Subclasses of light and heavy chains were determined with a mouse mAb isotyping kit (Amersham International). Other mAbs used in this work have already been described elsewhere2 7--30. Neutralization assays Neutralizing antibodies were measured with the in vitro rapid fluorescent focus inhibition test (RFFIT) 3t adapted to a 96-well microtitre plate a2. The technique originally designed with CVS was adapted to EBL1 as previously described la. The RFFIT standard was an HRIG obtained from Centre de Transfusion Sanguine, Nancy, France, diluted to contain 10 International units per ml (IUml-~). It contained 1.38 Equivalent Units per ml (EUm1-1) of EBLl-specific antibodies. Neutralizing titres were expressed in IU ml-~ against CVS and in EU ml- x against other viruses. Capacity of mAbs to neutralize a collection of viruses was evaluated by the in vitro neutralization test 33. Virus neutralizing index (NI) was determined by comparing titres of virus incubated with either medium or mAbs. Neutralization was considered positive when the NI was > 2 log~o units. Rabies immune globulins of human (HRIG) or equine (ERIG) origins ERIG with a neutralizing activity against CVS of

1260 Vaccine, Vol. 11, Issue 12, 1993

281 I U m l - t and of 115.5 EUm1-1 against EBL1, and HRIG containing 277 CVS IU m1-1 and 12.8 EBL1 EU ml- 1 were used in serotherapy experiments.

Neutralization-resistant variants Ten neutralization-resistant variants (RV 8-2) were selected and analysed as described previously33 using the PV strain of rabies virus and mAb 8-2. Resistant viruses RV 231-22 and RV 101-1 were described elsewhere 33. Competitive binding assays MAb 8-2 was biotinylated according to standard procedures and competition assays were performed as previously described 34. Binding of biotinylated mAb was evaluated with streptavidin-horseradish peroxidase. Percentage of competition was calculated using the formula [((Ama x - Atest)/Amax)x 100] where Areax is the absorbance read at 405 nm in wells containing 50 #1 of biotinylated mAb and 50/A of diluent and Atest is the absorbance in wells containing 50/zl of biotinylated mAb and 50 #1 of each different mAb. Sequence analysis BSR cells were infected with 1 p.f.u./cell of either PV or its neutralization-resistant variant RV 8-2 clone 4. Three days after infection, nucleic acids were extracted from infected cells, then rabies transcripts were amplified and sequenced as previously described 3s. Briefly, reverse transcription was carried out over the rabies virus genome using the oligodeoxynucleotide M2V ( + ) 5'-TTG GAA TAC TCT CAG GAG-3' and MMLV reverse transcriptase. The resulting RNA/cDNA hybrid was amplified by the polymerase chain reaction (PCR) using two oligodeoxynucleotides: M2V ( + ) and L ( - ) 5'-CAA AGG AGA GTT GAG ATT GTA GTC-3' and Taq polymerase in a Hybaid (Thermal Reactor) PCR apparatus. The amplification product was purified by electrophoresis in 0.8% Newsieve GTG agarose gel and then sequenced with the TTSequencing kit (Pharmacia LKB Biotechnology AB). Oligodeoxynucleotide primers derived from the sequence of the PV strain of rabies virus 36 and covering the whole G gene were used to prime sequence reaction. Protection studies Groups of 4-week-old female outbred OF1 Swiss mice (IFFA Credo) were inoculated intramuscularly (i.m.) in each hind leg with 100 #1 (1 x 107 p.f.u.) infectious virus (PV, EBL1 or CVS). Two hours later they were injected intraperitoneally (i.p.) with 200#1 mAbs or RIGs. Animals were observed for signs of disease or death and recorded daily. At the end of the test, paralytic mice, if any, were considered as not protected. Mortality of control mice was always between 75% (6/9) and 87.5% (7/8). Results were expressed as cumulative percentage of survivors. Percentage of protection was calculated using the formula [100x(dead control mice-dead treated mice)/dead control mice]. RESULTS

Characterization of mAb 8-2 Hybridoma 8-2 obtained after fusion of murine myeloma SP20 Ag with splenocytes of mice immunized with EBL1 secreted an IgG2= mAb with a x light chain. This mAb was strongly reactive in ELISA with purified

Rabies serotherapy in mice: J.A. Montar~o-Hirose e t

Neutralizing activity of a panel of anti-glycoprotein antibodies, including mAb 8-2, was tested with ten 8-2 escaping mutants (Table 2). According to their reactivity, the mutants could be arranged in three groups. In addition to the loss of sensitivity to mAb 8-2, mutants were not neutralized any more by mAb 231-22 recognizing site IIa and by mAb 110-3 of site IV. In contrast they were still neutralized by mAbs 101-1 and PVE which recognize site lib, by mAb 509-6 of site I and by mAbs 507-1, 718-4 of site III. These results indicated that glycoprotein mutations that modify neutralizing capacity of mAbs of site IIa and IV also modify mAb 8-2 neutralization. Reactivity of mAb 8-2 was tested on mutants already characterized and known to be affected respectively in sites I (RV 509-6), IIa (RV 231-22), lib (RV 101-1) and also with a mutant (RV 2-22C5) which seems to belong to several sites (II, III and VI) (Table 3). Only mutants affected in site IIa escaped mAb 8-2 neutralization. MAb 8-2 did not bind RV 8-2 infected cells in ELISA whereas it bound to cells infected with parental virus (data not shown), indicating that mutant RV 8-2 escapes neutralization because it is not recognized any more by the antibody. In order to localize the mutation that inhibits mAb binding and subsequent neutralization, amino acid substitutions in mutant glycoprotein were determined by sequence analysis.

EBL1 and recognized viral proteins on the surface of EBLl-infected fibroblasts as shown by immunofluorescence. It immunoprecipitated the glycoprotein (data not shown), suggesting that it is specific for an epitope located on the virus envelope glycoprotein. MAb 8-2 could not bind to immunoblotted and reduced glycoprotein, indicating that it recognizes a conformational epitope. MAb 8-2 exhibited a strong neutralizing activity of 524EUmg -1 towards EBL1 and all the representative bat isolates collected so far throughout Europe (Table 1). Neutralization activity of mAb 8-2 was assayed on several laboratory and wild isolates (Table 1). Unlike mAbs PVE or PVK, mAb 8-2 neutralized all the strains of rabies virus and EBL tested as well. In competition experiments, mAb PVE markedly inhibited the binding of mAb 8-2 whereas unrelated mAb (anti N-protein antibody) was ineffective (data not shown). Further characterization of the epitope recognized by mAb 8-2 was carried out with neutralization escaping mutants.

Characterization of neutralization escaping mutants Recent work 37 with neutralization escaping mutants and mAbs, extending previously described studies 3°'33, defined two major antigenic determinants (sites II and III), one minor site (site a), and several independent epitopes (sites I, IV, V and VI and epitope G1) for the rabies virus glycoprotein. Site II, the most antigenic site, can be divided into three subsites: IIa, IIb and IIc. In the present study, rabies variants escaping neutralization by mAb 8-2 were selected from a parental PV population after treatment with an excess of neutralizing antibody.

Table 1

al.

Sequence analysis The entire glycoprotein genes of mutant RV 8-2 clone 4 and of the parental virus strain (PV strain) were sequenced. Mutant glycoprotein sequence was found to differ from the parental sequence by a single amino

In vitro neutralization of laboratory and wild lyssavirus strains by monoclonal antibodies Monoclonal antibodies

Serotype 1 rabies

EBL1

EBL2

Virus

8-2

2-22C5

C1469

6-15C4

PVE

PVK

244

PM CVS R F F I T

+

+

+

+

--

+

-

+

+

+

+

-

+

-

C V S 113

+

+

+

+

+

+

-

ERA SAD

+ +

+ +

+ +

+ +

+ +

+ +

-

PAS PV

+ +

+ -

+ -

+ -t-

+ +

+

-

HEP

+

+

+

+

+

--

--

LEP YUG 333 fox

+ +

+ +

+ +

+ -

+ +

+ -

--

YUG 314 horse YUG 308 fox

+ +

+ +

+ +

+

+ +

-

-+

YUG 5 sheep

+

+

+

+

+

--

-

POL 283 RAC dog POL 247 RAC dog POL 270 RAC dog

+ + +

+ + +

+ + +

+ + +

+ + -

+ + -

-

P O L 101 f o x POL 9 9 fox

+

+

+

+

+

+

+

+

+

--

+

--

--

YUG 272 badger

+

--

-

-

FRG bat DAN bat SPA bat D D R bat F R A bat

+ + + + +

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

POL bat 85

+

.

.

.

.

.

.

POL bat 90

+

.

.

.

.

.

.

NETH bat R30 NETH bat R8

+ +

. .

. .

. .

. .

. .

. .

-

-

+

+ , Neutralization (NI >2); - , negative (NI <2). MAb 244 is a Mokola-derived neutralizing antibody. For virus strains see Materials and methods

V a c c i n e , Vol. 11, I s s u e 12, 1993

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Rabies serotherapy in mice: J.A. Monta~o-Hirose

e t al.

acid mutation at residue 198, resulting in lysine replacement by glutamate, confirming that epitope recognized by mAb 8-2 is located in site IIa on the glycoprotein. This mutation was already shown to affect RV 231-22 selected from CVS with mAb 231.2238'39.

Table 2 vi ruses

Pattern of neutralization of parental (PV) and of 8-2 resistant

8-2 R e s i s t a n t v i r u s e s Site

mAbs

PV

n= 4

I

509-6

+

+

+

+

Ila

231-22 220-8 8-2

+ . +

--

--

--

-. --

--

162-3 101-1 PVE

+ + +

-+ +

___ + +

+ + +

lib

.

n= 3

.

n= 3

1113-1

+

+

+

+

IIIb

507-1 718-4 1122-3

+ + +

+ + +

+ + ___

+ + +

IV

110-3

+

-

-

-

v

1118-6 1120-10

. .

vI

6-15C4

+

+

+

+

ND

1114-2 1106-2 904-4 1108-1 1119-8 2-22C5 PVK C1469

+ + ,+ + + + + +

+ + + _ + _+ +

+ + ,+ -t___ -t+

+ + ___ ,+ + +

. .

Table 4

. .

+ , Neutralization (NI > 2 ) ; --, negative (NI < 2 ) ; + , w e a k neutralization (NI = 2 ) . Parental virus is PV strain. ND, not d e t e r m i n e d

Table 3

Virulence of 8-2 resistant viruses after intracerebral inoculation Virulence of resistant mutant RV 8-2 clone 4 was tested in outbred mice inoculated intracerebrally (i.e.) and compared with parental virulence (Table 4). Equivalent quantitites (p.f.u.) of parent and mutant were inoculated. Inoculation with mutant virus resulted in an important reduced mortality rate compared with parental inoculation. As calculated from Table 4 by the method of Reed and Muench 4° 13 days after inoculation, 5 p.f.u, of parental virus were enough to kill 50% of the mice, whereas 600 p.f.u, of mutant were required to produce the same effect. Mutant infection was characterized by a longer incubation period, by hyperexcitation and squeaks and by a long-lasting ascendant paralysis from the hind legs towards the neck. Murine model for serotherapy The ability of RGIs or mAbs to protect mice passively against rabies virus or EBL1 was tested in mice injected

Ilia

. .

Since it was related to virulence attenuation39, virulence of 8-2 resistant variants was tested in mice.

Morbidity and mortality after 13 days of observation

Strain

D o s e (p.f.u.)

Dead o r s i c k / i n o c u l a t e d m i c e

PV

720 72 7.2 0.72 O.07 600 60 6 0.6 0.06

6/6 6/6 6/6 1/6 0/6 3a/6 0/6 0/6 0/6 0/6

RV 8-2

"Including o n e animal positive in the fluorescent antibody test, killed without signs of disease

Reactivity of s o m e neutralization-resistant viruses with a panel of neutralizing glycoprotein specific antibodies Virus variants

Site

mAbs

RV 509-6 (I)"

RV 231-22 (lla)"

RV 8-2 (lla)"

RV 101-1 (lib)"

RV 2-22C5 (ND)'

Ila

231-22 220-8 8-2

+ + +

----

---

--+

+ + -

lib

162-3 1116-1 1121-2 101-1 1111-1 PVE

+ + + + + -

. -+ + + --

+ + + + +

-----

+ + ---

IIc

1112-1 1105-11 1107-1 240-3 613-2

+ + + + +

+ +

+ + + +

+ + -

+ + + + +

VI

C-15C4

+

+

+

+

--

ND

PVK 2-22C5 C1469

+ + -

+ . -

--

+

+

-

-

+ , Neutralization (NI > 2 ) . • Site affected by mutation ND, not d e t e r m i n e d

1262

Vaccine,

- - , Negative (NI < 2 )

V o l . 11, I s s u e

12, 1 9 9 3

.

.

.

. +

.

.

Rabies serotherapy in mice: J.A. Monta~o-Hirose et al.

i.m. with virus. Mortality rates higher than 75% were obtained in outbred mice by i.m. inoculation of 107 p.f.u. of fixed rabies virus or EBL1. These infectious doses are markedly higher than those necessary to kill by the i.c. route. Death occurred 7-14 days after infection. Passive transfer of mAbs was performed i.p. The protective activity of variable amounts of antibodies injected at various time intervals after or before virus inoculation was tested. Mortality rates of mice inoculated with PV rabies virus strain and treated with homologous neutralizing monoclonal antibody (mAb 16-5) are presented in Table 5. Amounts of mAbs required to protect mice increased proportionally with the length of time between virus inoculation and mAb transfer. The optimal period of time for i.p. treatment with rabies

Kinetics of monoclonal antibody treatment (mAb 16-5) in lethally rabies (PV strain) infected mice

Table 5

Monoclonal antibody doses

(EU/mouse)

MAb treatment time

2h 2h 4h 24 h 48 h

before after after

after after

140

14

1.4

EDso

0/8 0/8 018 318 4/8

3/8 2/9 5/9 6/9 6/9

7/8 718 7/8 6/8 618

7.21 6.14 14.0 31.1 35.5

Results are presented as number of dead animals at day 16 over number of inoculated animals. Mortality in control (medium inoculated animals) was 7/9 EDso,number of EU/mouse needed to protect 50% of inoculated animals

neutralizing mAbs was 2 h either before or after i.m. inoculation of virus, when the EDs0 (efficacious dose expressed in EU/mouse to protect 50% of animals) was minimal (7.2 and 6.1, respectively). Thereafter, in all serotherapy experiments mAbs were inoculated 2 h after viral challenge.

Passive protection by RIGs against rabies virus or EBLI Two dilutions (11 or 2.2 IU) of HRIG and of ERIG were passively transferred i.p. to mice inoculated with rabies virus (Figure 1A) or EBL1 (Figure 1B). After 14 days, it was observed that 11 IU of HRIG and ERIG could protect 62.5 %0and 50% of mice respectively against rabies virus, whereas the same doses could protect only 25% of mice against EBL1. The weak RIG-mediated protection against EBL1 could be related to their low contents of EBL1 cross-specific antibodies, since 10 IU contained 4.6 EU specific EBL1 antibodies and HRIG 0.5 EU. Similar results were obtained with three other batches of HRIG. Passive protection by mAb 8-2 against rabies virus or EBL1 Mice received different doses of mAb 8-2 after inoculation with EBL1 (Fioure 2A) or PV rabies virus strain (Fioure 2B). Mice receiving 50 EU of mAb 8-2 were fully protected against EBL1 and 62.5% of mice were still protected with 2.5 EU ofmAb 8-2. Protective activity of mAb 8-2 was significantly lower (20 times less) against rabies virus (Figure 2B) since 62.5% protection was conferred by 50 EU of mAb. Similar experiments were

% survivors

% survivors

A

B

100%

100%

80%

80%

60%'

60%

40% -

40% e i

'0

-0

20% -

20% 'O

0% 4

I

I

J

J

i

5

6

7

8

9

Days

I

I

1 0 11

I

12

post-infection

i

i

i

J

1 3 1 4 1 5 1 6 1"1

0% 4

i

I

I

i

i

5

6

7

8

9

Days

l

I

1 0 11

l

i

I

-e-

•

I

1 2 1 3 1 4 1 5 1(] 1 7

post-infection

Figure 1 Survival curve in mice infected with (A) rabies (CVS) or (B) EBL1 with different concentrations of RIG ( ) or without ( - - - , control). Protection was calculated as described in text. O, Control; m, HRIG (11 lU); I-I, HRIG (2 IU); A, ERIG (11 IU); A , ERIG (2 lU)

Vaccine, Vol. 11, Issue 12, 1993

1263

Rabies serotherapy in mice: J.A. Monta~o-Hirose et al. % survivors

% survivors

A

B

100%

100%

80%

80% i

I

60%

60% i t J

40%

40%

i

1I

4- Control 1 "Jr 8.2 50EU

20%

4"8.2 25 EU

-O-Control

20%

0

•

"=" 8.2 50 EU

-0

"e'8.2 3,5 EU

.0-

-o

"4" 8.2 2.6 EU

0%

0% 4

5

6

7

8

9

10 11

12

13 14 15

4

5

6

Days p o s t - i n o c u l a t i o n

7

8

10

9

11

12 13

14 15

Days p o s t - i n o c u l a t i o n

% survivors

% survivors

C

D

100%

100%

80%

80%

60%

60%

40%

40%

--

- -

.

--_

t

20%

"0-

20%

0. .o-

"~" 8.2 25 EU

•4P C o n t r o l

-~" PVE

"e . X L . . . ~

U.

"~" PVE 25 EU

.4- 26-9

0-

0

"m" PVE 10 EU

0%

0% 4

5

6

7

8

9

10

11

12

Days post-inoculation

13

14

15

4

5

6

7

8

9

10 11

12 13 14

Days post-inoculation

Figure 2 Survival curve in mice infected with EBL1 (A and C) or rabies virus PV strain (B and D) with different concentrations of mAbs ( ) or without (. . . . , controls). MAb 8-2 neutralizes EBL1 and PV strains. MAb PVE-3 neutralizes PV only and was used at 40 EU (C). MAb 26-9 is an unrelated non-neutralizing Mokola nucleocapsid-specific antibody

1264

Vaccine, VoI. 11, Issue 12, 1993

Rabies serotherapy in mice: J.A. Montar~o-Hirose et al.

performed with a PV-specific neutralizing mAb, PVE-3, which neutralizes PV rabies strain and not EBL1 (Figures 2C and 2D). This mAb could protect mice against PV (Figure 2D) but not against EBL1 (Figure 2C). Alteration of the EBL1 survival curve with PVE-3 was not significantly different from the variation observed with an unrelated non-neutralizing mAb, mAb 26-9, a Mokola nucleocapsid specific antibody (Figure 2C). This result indicates that antibody protection is related to the intrinsic neutralizing capacity of each monoclonal and excludes the possibility that an unspecific mechanism is involved in our serotherapy model.

Passive protection by RIG supplemented with mAb 8-2 against EBL1 Since RIG protected mice poorly against a peripheral inoculation of EBL1, in contrast to mAb 8-2, protective activity of a combination of RIG and mAb was tested (Figure 3). Injected alone, 11 IU of HRIG protected 12.5% of mice and 2.5 EU of mAb 8-2 protected 50% of animals. When mixed and injected together, 75% of mice were protected (Figure 3A). Protective activity of the mixture was the sum of the protective activities of both products. Similar results were obtained with ERIG:' 11 EU of ERIG protected 50% of animals and 1.25 EU of mAb 8-2 protected 12.5 %oof animals. The combination resulted in 62.5% protected animals (Figure 3B). % survivors

A 100% 80% 6o%

I

I

I

=

_-

_-

40% 20% 4k-

0%

4

I 5

I 6

I 7

I

8

I

9

I

10

1

- 41'-

ll

-O-

I

-O-

I

12

I

13

-O

-

5

14

1

O-

- O-

1 4'

1 '5

-6

'

16

17

D a y s p o s t - i n o c u l a t i on % survivors

B 100% 80% 6o% 40% 20% O

0%

4

5'

6'

7'

8' Days

9'

10

1'1

-

"O"

1 9'

-

"O-

1 3'

-

-O

1 '6

17

post-Inoculation

Figure 3 Survival curve of mice infected with EBL1 with a combination of different RIG and mAb 8-2 concentrations ( ) or without ( - - - , control). Mice were passively given HRIG (A) or ERIG (B). O, Control; *, RIG (11 lU); l , mAb 8-2 (2.5EU); A , mAb 8-2 (1.25EU); ,,, RIG (11 IU)+ mAb 8-2 (2.5 EU); +, RIG (11 IU)+ mAb 8-2 (1.25 EU)

In contrast, a combination of non-protective dilutions of RIG and mAb (1.25 EU of mAb and 11 IU of HRIG that individually protected 12.5 %0 of animals) resulted in a protection of 75% (Figure 3A). These results suggest that synergy exists between antibodies of different affinity but only at low antibody concentrations. DISCUSSION Persons who have been bitten by animals suspected or proven to be rabid, or by wild animals - including bats - are recommended to receive a specific postexposure treatment that consists of local infltration of rabies immune globulin in addition to the first vaccine dose. Variable content of EBLl-specific antibodies in HRIG addressed the question of whether it is necessary to adjust HRIG content for treatment of patients exposed to EBL 1 risk or to replace HRIG by EBLl-specific immune globulin preparations. The absence of vaccines prepared with EBL strains makes the preparation of EBL immunoglobulins of human origin difficult. Murine mAbs specific for EBL could be an alternative. Murine mAbs have been extensively used for human immunotherapy of carcinoma. The low incidence of severe allergic reactions associated with this immunotherapy9 indicates that murine mAbs would be well tolerated, especially in the case of only one injection as required for rabies prophylaxis. This paper describes the characterization of the murine mAb 8-2 generated against EBL1 and its use in the postexposure treatment of EBL1 in a mouse model. This mAb recognized an epitope located in the antigenic site IIa of rabies glycoprotein, as shown by the results of reactivity with a number of well characterized mutants and those of sequence analysis. This mutation seems to affect site IV (defined by mAb 110-3) as well as site IIa, because mAb 110-3 cannot neutralize the RV 8-2 further. As escaping mutants corresponding to this mAb are not available for crossreaction studies, it is not possible to assert that sites IV and II are overlapping. Passive protection by this mAb was tested in mice injected i.m. with virus and 2 h later with mAb administrated i.p. The i.m. route for virus injection was chosen to mimic rabies contamination, which mainly occurs through bites. Protective activity of mAb 8-2 was assayed in the absence of vaccine in order to measure the specific capacity of mAbs to prevent the infection. This mAb was protective not only against an EBL challenge but also against serologically unrelated viruses (CVS or PV rabies virus strains) and should be protective in vivo to many strains of various geographical origins in view of its lzroad spectrum of in vitro neutralizing reactivity. Because of the existence of antigenic variants and their possible induction in vivo under mAbs pressure 33, it is generally recommended that a neutralizing mAb should not be used alone but instead in a cocktail of functionally complementary mAbs (or added to current immune globulins). Nevertheless, in vitro neutralization escaping mutants selected after treatment of parental strains with an excess of mAb 8-2 were much less virulent for adult mice than parental virus. These data suggest that putative risk of an in vivo emergence of fully pathogenic antigenic mutants under pressure of this particular mAb could indeed be minimal.

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Owing to its large neutralizing spectrum and its eventual capacity to induce low pathogenic escaping mutants, the murine mAb 8-2 is a serious candidate for serotherapy against wild-life isolates including bat lyssaviruses. ACKNOWLEDGEMENTS The authors are grateful to Bernhard Dietzschold (The Wistar Institute, Philadelphia, PA, USA) for providing a panel of anti-G mAbs, to Hans Bunschoten and Albert Osterhaus (National Institute of Public Health, The Netherlands) for providing mAbs 2-22C5, C1469 and 6-15C4, to Ivan Vodopija (Zagreb Institute of Public Health, Zagreb, Croatia) and to Danuta Seroka (National Institute of Hygiene, Warsaw, Poland) for the wild rabies isolates. J.A.M.-H., from Instituto de Tecnologia do Paranh (TECPAR) (Curitiba, PR, Brazil), is the recipient of Brazilian fellowship 260116/89.0 from Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq).

REFERENCES 1

2 3 4 5

6

7 8

9 10 11 12 13

14

15

Koprowski, H. and Black, J. Studies on chick-embryo-adapted rabies virus. V. Protection of animals with antiserum and living attenuated virus after exposure to street strain of rabies virus. J. Immunol. 1954, 72, 85-93 Blancou, J., Soria Baltazar, R., Molli, I. and Stoltz, J.F. Effective postexposure treatment of rabies infected sheep with rabies immune globulin and vaccine. Vaccine 1991, 9, 432-437 Baltazard, M. and Bahmanyar, M. Essai pratique du s~rum antirabique chez les mordus par Ioups enrag(~s. Bull. WHO 1955, 13, 747-772 Wilde, H., Chomchey, P., Prakongsri, S., Puyaratabanchu, P. and Chutivongue, S. Adverse effects of equine rabies immune globulin. Vaccine 1989, 7, 10-11 Schumacher, C.L., Dietzschold, B., Ertl, H.C.J., Niu, H.S., Rupprecht, C.E. and Koprowski, H. Use of mouse antirabies moncclonal antibodies in postexposure treatment of rabies. J. Clln. Invest. 1989, 84, 971-975 Dietzschold, B., Gore, M., Casali, P., Ueki, Y., Rupprecht, C.E., Notkins, A.L. and Koprowski, H. Biological characterization of human monoclonal antibodies to rabies virus. J. Virol. 1990, 64(6), 3087-3090 Lafon, M., Edelman, L., Bouvet, J.P., Lafage, M. and Montch:~tre, E. Human monoclonal antibodies specific for the rabies virus glycoprotein and N protein. J. Gen. Virol. 1990, 71, 1689-1698 Enssle, K., Kurrle, R., K6hler, R., M(~ller, H., Kanzy, E.J., Hilfenhaus, J. and Seller, F.R. A rabies-specific human monoclonal antibody that protects mice against lethal rabies. Hybridoma 1991, 10(5), 547-556 Sears, H., Herlyn, D., Steplewski, Z. and Koprowski, H. Effects of monoclonal antibody immunotherapy on patients with gastrointestinal adenocarcinoma. J. Biol. Responses 1984, 3, 138-150 Kappeler, A. Bat rabies surveillance in Europe. WHO Rabies Bull. 1989, 13, 12-13 King, A., Davies, P. and Lawrie, A. The rabies viruses of bats. Vet. Microbiol. 1990, 23, 165-174 Lumio, J., Hillborn, M., Roine, R., Ketonen, L., Halthia, M., Valle, M. et al. Human rabies of bat origin in Europe. Lancet 1966, I, 378 Selimov, M.A., Tatarov, A.G., Botvinkin, A.D., Klueva, E.V., Kulikova, L.G. and Khismatullina, N.A. Rabies-related Yuli virus: identification with a panel of monoclonal antibodies. Acta Virol. (Praha) 1989, 33, 542-545 Dietzschold, B., Rupprecht, C., Lafon, M., Tollis, M., Mattei, J., Wiktor, T.J. and Koprowski, H. Antigenic diversity of the glycoprotein and nucleocapsid proteins of rabies and rabies-related virus. Implications for epidemiology and control of rabies. Rev. Infect. Dis. 1988, 10, $785-$798 Bourhy, H., Kissi, B., Lafon, M., Sacramento, D. and Tordo, N. Antigenic and molecular characterization of bat rabies virus in Europe. J. Clln. Microbiol. 1992, 30(9), 2419-2426

1266

Vaccine, Vol. 11, Issue 12, 1993

16 Schneider, L.G. Antigenic variants of rabies virus. Comp./mmuno/. Microbiol. Infect. Dis. 1982, S, 101-107 17 Dietzschold, B. Antigenic variation in rabies and rabies-relatad viruses: cross-protection independent of glycoprotein-mediated virus-neutralizing antibody. J. Infect. Dis. 1987, 156(5), 815-822 18 Lafon, M., Bourhy, H. and Sureau, P. Immunity against the European bat rabies (Duvenhage) virus induced by rabies vaccines: an experimental study in mice. Vaccine 1988, 6, 362-368 19 Lafon, M., Herzog, M. and Sureau, P. Human rabies vaccines induce neutralizing antibodies against the European bat rabies virus (Duvenhage). Lancet 1986, II, 515 20 Schneider, L.G., Mueller, W.W. and Hohnsbeen, K.P. Present human rabies vaccines and neutralizing antibody activity against the bat-rabies strain Duvenhage recently isolated in Poland, Denmark and the Federal Republic of Germany. WHO Rabies Bull. Eur. 1986, 2, 9-11 21 Cells, E., Ou, D., Dietzschold, B. and Koprowski, H. Recognition of rabies and rabies-related viruses by T cells derived from human vaccine recipients. J. Virol. 1988, 62, 3128-3134 22 Herzog, M., Fritzell, C., Lafage, M., Montafio-Hirose, J.A., Scott-Algara, D. and Lafon, M. T and B cell human responses to European bat lyssavirus after post-exposura rabies vaccination. C/in. Exp. Immunol. 1991, 85, 224-230 23 Sato, M., Maida, N., Yoshida, H., Urade, M., Saito, S., Miyazaki, T. et al. Plaque formation of Herpes virus hominidis type 2 and Rubella virus in variants isolated from the colonies of BHK21/WI-2 cells formed in soft agar. Arch. Virol. 1977, 53, 269-273 24 Petrovic, M Urban and sylvatic rabies in Yugoslavia. WHO Rabies Bull. Eur. 1987, 4, 16-18 25 Montafio-Hirose, J.A., Bourhy, H. and Lafon, M. A reduced panel of anti-nucleocapsid monoclonal antibodies for bat rabies virus identification in Europe. Res Virol. 1990, 141,571-581 26 Libeau, G. and Lafon, M. Production of monoclonal antibodies against the Pasteur (P.V.) strain of rabies virus. Dev. Biol. Stand. 1984, 57, 213-218 27 Wiktor, T.J. and Koprowski, H. Monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants. Prcc. Acad. Sci. USA 1978, 75, 3938-3942 28 Wiktor, T.J. and Koprowski, H. Antigenic variants of rabies virus. J. Exp. Med. 1980, 152, 99-112 29 Libeau, G., Lafon, M. and Rollin, P. Etude de la sp6cificit(~ d'anticorps monoclonaux obtenus avec la souche de virus de la rage Pasteur PV. Rev. Elev. M~cl. Vet. Pays Trop. 1964, 374, 383-394 30 Bunschoten, H., Gore, M., Claasen, I.J.T.M., Uytdehaag, F.G.C.M., Dietzschold, B., Wunner, W. and Osterhaus, A.D.M.E. Characterization of a new virus-neutralizing epitope that denotes a sequential determinant on the rabies virus glycoprotein. J. Gen. Virol. 1989, 70, 291-298 31 Smith, J.S., Yager, P.A. and Baer, G.M. A rapid tissue culture test for determining rabies neutralizing antibody. In: Laboratory Techniques in Rabies (Eds Kaplan, M.M. and Koprowski, H.) 2nd Edn, WHO Monograph Series no. 23, WHO, Geneva, pp. 354-357 32 Zalen, E., Wilson, C. and Pukitis, D. A microtest for the quantitation of rabies virus neutralizing antibodies. J. Biol. Stand. 1979, 7, 213-220 33 Lafon, M., Wiktor, T.J. and Macfarlan, R.I. Antigenic sites on the CVS rabies virus glycoprotein: analysis with monoclonal antibodies. J. Gen. Virol. 1983, 64, 843-851 34 Lafon, M. and Wiktor, T.J. Antigen sites on the ERA rabies virus nucleoprotein and non-structural protein. J. Gen. Virol. 1985, 56, 2125-2133 35 Sacramento, D., Bourhy, H. and Tordo, N. PCR technique as an alternative method for diagnosis and molecular epidemiology of rabies virus. Mol. Cell. Probes 1991, 5, 229-240 36 Tordo, N., Poch, O., Ermine, A., Keith, G. and Rougeon, F. Walking along the rabies genome: is the large G-L intergenic region a remnant gene? Prec. Natl. Acad. Sci. USA 1966, 83, 3914-3918 37 Benmansour, A., Leblois, H., Coulon, P., Tuffereau, C., Gaudin, Y., Flamand, A. and Lafay, F. AnUgenicity of rabies virus glycoprotein. J. Virol. 1991, 65(8), 4198-4203 38 Wunner, W.H., Dietzschold, B., Smith, C.L., Lafon, M. and Golub, E. Antigenic variants of CVS rabies virus with altered glycosylation sites. Virology 1985, 140, 1-12 39 Prehaud, C., Coulon, P., Lafay, F., Thiers, C. and Flamand, A. Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence. J. Virol. 1988, 62, 1-7 40 Lorenz, R.J. and B0gel, K. Methods of calculation. In: Laboratory Technique in Rabies (Eds Kaplan, M.M. and Koprowski, H.) 2nd Edn, WHO Monograph Series no. 23, WHO, Geneva, pp. 321-335