Novel monoclonal antibodies that bind to wild and fixed rabies virus strains

Journal of Virological Methods 175 (2011) 66–73

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Novel monoclonal antibodies that bind to wild and fixed rabies virus strains Camila Zanluca a , Luan Renato dos Passos Aires a , Paula Pazzini Mueller a , Vanessa Valgas dos Santos a , Maria Luiza Carrieri b , Aguinaldo Roberto Pinto a , Carlos Roberto Zanetti a,∗ a b

Laboratório de Imunologia Aplicada, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina, 88040-970, Florianópolis, SC, Brazil Instituto Pasteur, Avenida Paulista 393, 01311-000, São Paulo, SP, Brazil

a b s t r a c t Article history: Received 23 November 2010 Received in revised form 12 April 2011 Accepted 19 April 2011 Available online 28 April 2011 Keywords: Rabies Monoclonal antibody Brazilian wild rabies isolates Fixed rabies virus strains

Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3␬, IgG2a␬, IgM␬, and an IgG2b␬ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Despite being known for more than three millennia, rabies, a fatal encephalomyelitis, still infects many humans, as well as, many domestic and wild animals around the world. The disease is caused by the rabies virus, a single-stranded RNA virus belonging to the Rhabdoviridae family, genus Lyssavirus (Warrel and Warrel, 2004). The virion is composed of two structural units, the ribonucleoprotein (RNP) and an envelope. RNP is formed by RNA and three viral proteins, namely the nucleoprotein, the phosphoprotein, and the RNA-dependent RNA polymerase. The envelope consists of glycoproteins anchored to a host-derived lipid bilayer surrounding the matrix protein. The glycoprotein allows virus attachment to cellular receptors, mediates membrane fusion, and serves as a target for virus-neutralizing antibodies (Rupprecht et al., 2002; Schnell et al., 2010). The first monoclonal antibodies (MAbs) against the rabies virus were produced by Wiktor and Koprowski (1978) and initially were employed for the detection of antigenic variants among several strains of rabies virus. Anti-RNP and anti-glycoprotein rabies MAbs have been used to characterize some strains of rabies virus and to distinguish rabies from other rabies-related viruses (Flamand et al., 1980a,b). MAbs also proved to be important tools for diagnosis and immunoassays (Lembo et al., 2006; Zhang et al., 2009), and

∗ Corresponding author. Tel.: +55 48 37215206; fax: +55 48 37219258. E-mail address: [email protected] (C.R. Zanetti). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.04.019

the protective activity of MAbs are being investigated currently as a possible post-exposure treatment (Bakker et al., 2008; Müller et al., 2009). Antigenic analysis of rabies isolates in Latin America is carried out mainly by using a panel of eight MAbs established by Diaz et al. (1994) and provided by the Centers for Disease Control and Prevention (Atlanta, USA). This panel of MAbs helps to identify a characteristic antigenic make up in rabies viruses circulating in stable enzootics within a specific species of carnivore or bat. Thus, by identifying the rabies virus antigenic pattern, the most likely rabies transmitter or rabies reservoir can be inferred. However, as a consequence of improved rabies surveillance programs and the natural evolution of rabies viruses in Brazil and several other countries of the Americas, new antigenic variants have been identified and possibly are associated with different species of carnivores and bats. These new antigenic variants have resulted in the restricted use of the current MAb panel to identify potentially emergent rabies reservoirs. Thus, although a large number of anti-rabies MAbs were reported in the last few years, a demand exists for the production of new MAbs capable of detecting variants which are incompatible to the panel used in Brazil. In addition, rabies conjugates for diagnostic use in Brazil are obtained mainly from the serum samples of immunized animals. The use of MAbs could increase the specificity of rabies conjugates (Woldehiwet, 2005). The production of MAbs also attracts investment in simple and fast diagnostic tests to evaluate infection in field conditions. Finally, the test most widely employed to determine the strength of the rabies vaccine is the NIH test,

C. Zanluca et al. / Journal of Virological Methods 175 (2011) 66–73

which presents several problems in terms of reproducibility. The results from this test do not have a satisfactory correlation with the formation of virus-neutralizing antibodies (Barth et al., 1988). Since the rabies glycoprotein induces neutralizing antibodies and protection, the standardization of an immunoassay using antiglycoprotein MAbs could be researched as a possible replacement for the NIH test in determining rabies vaccine strength. Considering these possible uses for rabies MAbs, the present study reports the production of ten MAbs, including a virus-neutralizing MAb, that react against wild rabies isolates and fixed rabies strains. The perspective of further applications for these MAbs will be discussed. 2. Animals and methods 2.1. Animals Male Balb/c mice (30- to 45-days-old, mean weight of 20 g) were used in the immunization protocols. Animals were maintained at the Animal Facility of the Microbiology, Immunology, and Parasitology Department at the Federal University of Santa Catarina. Animal procedures were approved by the Federal University of Santa Catarina Ethics Committee on Animal Research. 2.2. Cell lines Mouse myeloma cell line P3X63Ag8.653 (ATCC CRL-1580) and hybridomas were maintained in RPMI-1640 medium (Cultilab, Campinas, Brazil) and supplemented with 20% fetal bovine serum (FBS – Cultilab), 23.8 mM sodium bicarbonate, 0.2 mM l-glutamine, 1.0 mM sodium pyruvate, 9.6 mM HEPES, and antibiotics (100 IU/ml penicillin, 100 ␮g/ml streptomycin, and 0.25 ␮g/ml amphotericin B – Sigma–Aldrich, St. Louis, USA) at 37 ◦ C, 5% CO2 atmosphere. Baby hamster kidney cells (BHK-21, ATCC CCL-10) and N2A cells (ATCC CCL-131) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Ham F12 (DMEM F12 – Cultilab) with 10% FBS for BHK-21 and 20% FBS for N2A cells, 14.0 mM sodium bicarbonate and antibiotics at 37 ◦ C, 5% CO2 atmosphere. S2MtRVGP-Hy cells (Lemos et al., 2009) were cultured in SF-900 II SFM medium (Invitrogen, Grand Island, USA) and maintained at 28 ◦ C.

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on days 7, 14, and 36 without adjuvant. Nineteen days later, mice were challenged with the respective virus isolates inoculated by footpad injection, followed by boosting with inactivated suspension on day 72. In the fifth protocol, three mice received four doses of a recombinant fragment of the rabies virus glycoprotein, rGERA179–281 (Bassi et al., 2008). Doses containing 40 ␮g of the protein were administered s.c. on day 0 in complete Freund’s adjuvant, on day 7 in incomplete Freund’s adjuvant, and i.p. on days 14 and 28 without any adjuvant. Mice were boosted further with live PV rabies virus strain (∼64 CCID50% ) by footpad injection on day 34. 2.4. Monoclonal antibody production Three days after the last immunization or eight days after the boost (protocol three), mice were humanely killed and their spleens were collected aseptically. Cells were released by applying gentle pressure to the capsule of the organ and placing it between two glass slides, and then the content was filtered in nylon membranes. Red blood cells were lysed using 5 ml of cold lysis solution (168.08 mM ammonium chloride, 9.98 mM potassium bicarbonate, 0.09 mM EDTA, pH 7.4). After two washes with RPMI-1640 medium, splenocytes were fused with P3X63Ag8.653 myeloma cells using polyethylene glycol 4000 (PEG – Sigma–Aldrich) as the fusion agent. The fusion was performed by adding PEG 50% in RPMI1640 medium without FBS for 2 min, followed by washing with RPMI-1640 medium for 5 min, as described previously (Yokoyama et al., 2006). Hybrid cells were selected by growth in RPMI-1640 with the supplements described above plus 0.1 mM hypoxanthine, 0.0004 mM aminopterine, and 0.016 mM thymidine (HAT medium) from 24 h after fusion until the 14th day. The hybridoma supernatants were screened by indirect immunofluorescence (IIF) as described below. Hybridomas whose supernatants showed positive results by the screening tests were subjected to two rounds of the limiting dilution method and stored in liquid nitrogen. The immunoglobulin isotypes of the MAbs were determined using an SBA ClonotypingTM System/HRP (Southern Biotech, Birmingham, USA), following manufacturer instructions.

2.3. Animal immunization

2.5. Screening test

Five immunization protocols were carried out. In the first protocol, three Balb/c mice were immunized four times intraperitoneally (i.p.) with 500 ␮l of human rabies vaccine (VeroRabTM – Aventis Pasteur, Lyon, France) diluted 1:10 in PBS on days 0, 7, 14, and 28. In the second protocol, two mice were immunized with wild rabies virus isolated from a bovine infected naturally by a vampire bat in Passos de Torres, in the state of Santa Catarina, Brazil. Mice were immunized subcutaneously (s.c.) with 500 ␮l of UV lightinactivated 10% infected mouse brain suspension (∼320 cell culture infectious dose 50% – CCID50% ), mixed with complete Freund’s adjuvant (Sigma–Aldrich). Seven days later, a second subcutaneous immunization was performed using the same amount of antigen mixed with incomplete Freund’s adjuvant (Sigma–Aldrich). Two additional doses were given i.p. at 7-day intervals using the same antigen dose, followed by a fifth i.p. dose one month later. In two other protocols, mice were immunized with wild rabies viruses isolated from Nyctinomops laticaudatus (Nl) and Eptesicus furinalis (Ef) bats. Four Balb/c mice received four 500 ␮l doses of UV light-inactivated 20% infected mouse brain suspensions diluted 1:5 in PBS, two mice received the Nl virus (∼20 CCID50% ), and the remaining two mice received the Ef virus (∼80 CCID50% ). Doses were administered s.c on day 0 with complete Freund’s adjuvant, and i.p.

Hybridomas secreting antibodies against rabies virus were selected by IIF in rabies virus PV strain-infected BHK-21 cells and in control uninfected BHK-21 cells. Cells were fixed at 24 h post-infection with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, both steps for 20 min at room temperature. Hybridoma supernatants were added as first antibodies and incubated for 30 min at 37 ◦ C. To detect reactive monoclonal antibodies, incubation was performed for 1 h at 37 ◦ C with FITC-conjugated anti-mouse immunoglobulins (Sigma–Aldrich). An Olympus IX51 microscope connected to a digital camera (Olympus DP72, Center Valley, USA) was used to record the results. Sera from rabies virus-immunized mice and non-correlated MAbs that bind IMNV, a virus that infects marine invertebrates, were used as positive and negative controls, respectively. In addition, when mice were immunized with wild rabies virus, hybridomas were screened by IIF on BHK-21cells infected with the homologous virus. rGERA179–281 is a fragment of the glycoprotein from rabies virus ERA strain and shows 98% of similarity to rabies virus PV strain (Bassi et al., 2008). Hence, for practical issues, in the fifth immunization protocol, when mice were immunized with this recombinant protein followed by a boost with rabies PV strain, the only screening test performed was IIF on rabies virus PV straininfected BHK-21 cells.

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2.6. Neutralization activity of MAbs

2.10. Reactivity of MAbs to wild rabies virus isolates

The rabies-specific virus-neutralizing activity of MAbs was determined by the simplified fluorescence inhibition microtest as described by Favoretto et al. (1993). Briefly, serial two-fold dilutions of hybridoma supernatants were incubated with a rabies virus PV strain suspension at a previously established amount, enough to infect 90–100% of cells, for 1 h at 37 ◦ C. Next, the mixture was used to infect 5 × 104 BHK-21 cells. After 24 h incubation at 37 ◦ C in a 5% CO2 atmosphere, the cells were fixed with paraformaldehyde 4% and submitted to IIF as described above. Virus neutralization was determined by comparing the number of infected cells in cultures incubated with antibody-treated virus to the number of infected cells in culture incubated with untreated virus.

Reactivity of the MAbs against Brazilian rabies virus isolates (Table 1) was determined using the indirect immunofluorescent technique, carried out according to Diaz et al. (1994). Briefly, slides with the imprint of central nervous system tissues from mice infected with wild virus isolates were prepared. Tissues were fixed overnight in acetone at −20 ◦ C and overlaid with the supernatant of hybridomas for 30 min at 37 ◦ C. Next, individual slide impressions were rinsed carefully with PBS, followed by the addition of antimouse polyvalent FITC-conjugated immunoglobulins for 30 min at 37 ◦ C. After two washes with PBS and one wash with distilled water, reactions were visualized using a UV light microscope. The antigenic variants of the isolates were determined using the panel of MAbs provided by CDC as well.

2.7. Reaction to rGERA179–281 To detect antibodies that react against rGERA179–281, MAbs were tested by ELISA, using a protocol adapted from Piza et al. (1999). Briefly, 96-well plates (Costar-3590, Corning, New York, USA) were coated with rGERA179–281, 200 ng/well in 0.05M sodium carbonate–bicarbonate buffer (pH 9.6) overnight at 4 ◦ C. All further incubation steps were performed for 1 h at 37 ◦ C, unless stated otherwise. Non-specific binding was blocked with 0.5% (w/v) gelatin in Tris–NaCl buffer (50 mM Tris, 150 mM NaCl, pH 9.6) for 30 min. MAbs were loaded onto the ELISA plate and binding was detected using anti-mouse polyvalent immunoglobulins labeled with peroxidase (Sigma–Aldrich). Sera from rabies-immunized mice and cell-free hybridoma culture medium were used as positive and negative controls, respectively. Between each step of the reaction, microplates were washed five times with phosphate buffered saline (PBS, pH 7.2) containing 0.05% Tween 20. Reactions were developed with o-fenilenodiamine (OPD) in the presence of H2 O2 , with absorbance measured at 492 nm using a SunriseTM Basic system (Tecan, Männedorf, Switzerland). 2.8. Reactivity against rabies virus glycoprotein For rabies virus glycoprotein expression, S2MtRVGP-Hy cells were cultured as previously described (Lemos et al., 2009). Glycoprotein expression was induced by 700 ␮M of CuSO4 for 72 h. 2 × 105 cells/well were added to a 96-well plate. After adhesion, cells were fixed with 80% acetone for 30 min at 4 ◦ C. Antiglycoprotein MAbs were detected by incubating the cells with supernatants from hybridomas for 30 min at 37 ◦ C, followed by FITC-conjugated anti-mouse immunoglobulins for 1 h at 37 ◦ C. As positive and negative controls, sera from rabies-immunized mice and a non-relating MAb against IMNV were used, respectively. 2.9. Reactivity of MAbs against inactivated rabies virus present in commercial vaccines Human and veterinary rabies vaccines were applied to nitrocellulose membranes (GE-Healthcare, Little Chalfont, UK) at a volume of 2 ␮l/spot to perform an immunodot-blot assay. All further incubation steps were carried out for 1 h at room temperature. Membranes were blocked with 5% non-fat milk in TBS-T (20 mM Tris, 137 mM NaCl, pH 7.6, containing 0.1% Tween 20) and incubated with hybridoma supernatants, using supernatants containing a non-relating MAb against IMNV as the negative control. Bound antibodies were detected using anti-mouse polyvalent immunoglobulins conjugated with peroxidase (Sigma–Aldrich). ECL Western blotting detection reagents (AmershamTM , Piscataway, USA) were used to detect the signals.

3. Results 3.1. Production and characterization of monoclonal antibodies against rabies virus Five fusion protocols were carried out. In the first four protocols, mice were immunized with rabies vaccine or an inactivated wild rabies virus. Six stable hybridomas, which produce antibodies that bind to rabies virus on IIF, were obtained (Fig. 1A–F). For protocols in which mice were immunized with wild rabies virus, the same results were obtained for the IIF screening of BHK-21 cells infected with the PV strain of rabies virus as were obtained for the homologous virus. However, only the results obtained using the former virus were shown. Since one of the objectives of this work was to produce neutralizing MAbs, a new strategy for immunization was achieved. Accordingly, in the fifth fusion protocol, mice were immunized with rGERA179–281, followed by boost with an infective rabies strain to maximize the specific response to the virus. In this protocol, four hybrid cells had positive IIF results (Fig. 1G–J), with one producing neutralizing antibodies (Table 2). The staining patterns of MAbs on IIF were distinct, as can be seen in Fig. 1. The positive control and all but one MAb showed the well-circumscribed intracytoplasmic inclusions characteristic of anti-nucleocapside MAbs, while the neutralizing MAb (8D11) showed diffuse staining, predominantly in the cell membrane. The MAbs showed no cross-reaction with non-infected BHK-21 cells (data not shown). Isotyping of these MAbs revealed four MAbs were IgG3, three were IgG2a, two were IgM, and one was IgG2b. All ten revealed kappa light chains as well (Table 2). Only MAb 7B7 recognized rGERA179–281 (Table 2). MAbs secreted by hybridomas originating from the fifth protocol did not recognize the recombinant protein with which mice were immunized. This lack of protein recognition was most likely due to the strong immune response generated against other epitopes of rabies virus. Nonimmunized mice, which only received the virus, also produced a similar immune response (data not shown). In order to identify specific MAbs against the rabies glycoprotein, IIF assays were carried out on S2MtRVGP-Hy cells, a Drosophila melanogaster Schneider 2 cell line expressing whole rabies virus glycoprotein. Only MAb 8D11, which also was able to neutralize the virus, showed a positive result in this test, confirming its specificity against the viral glycoprotein (Fig. 2). Surprisingly, MAb 7B7, which was able to react against the rGERA179–281 protein, did not bind to the glycoprotein expressed by S2MtRVGP-Hy cells. No reaction was observed when anti-IMNV MAb was used as the negative control (data not shown).

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Table 1 Immunoreactivity of monoclonal antibodies against Brazilian rabies virus isolates on indirect immunofluorescence assay. Sample numbers CVS 1023 1024 1026 1027 1032 2429 6292 6462 6879 7062 7063 1028 6746 7488 3176 4607 4616 4697 5610 5787 5861 6207 6375 6429 6945 7279 7952 1017 1043 5634 5636 1016 4194 5635 6634

Host

LIA 02

6H8

7B7

3E6

8D11

9C7

8B6

Antigenic variant

Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Equine Equine Human Bat Bat Bat Bat Bat Bat Bat Bat Bat Bat Bat Bat Bat Fox Fox Fox Fox Dog Dog Dog Dog

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ − ++ ++ ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

− − − − − − − ++ − + + + − − − − − − − ++ − + − − − − − − − − − − − − − −

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ − ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ − ++ ++ ++ + ++ ++ ++ ++

− + ++ + ++ ++ ++ − − ++ − − − − − − + − − ++ − + + − + − − − − − − − − − − −

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ − ++ ++ + ++ ++ ++ + ++ ++ ++ ++

++ − − − − − − − − + − − − − ++ − + ++ ++ ++ + ++ − + − ++ + − ++ ++ ++ ++ ++ ++ ++ −

Laboratory strain 3* 3* 3* 3* 3* 3 3 3 3 3 3 3* 3 2 4 3 4 NC 3 4 3 3 4 4 NC 3 NC 2 2 2 2 2 NC 2 3*

CVS: challenge virus standard, used as positive control; −: negative; +: weakly positive; ++: positive; NC: variant not compatible with the profiles defined by the panel of MAbs provided by CDC; 3* isolates characterized as variant 3, except for an antibody of the panel.

3.2. Reactivity of MAbs against wild rabies virus isolates Aiming to investigate the possibility of using the MAbs for diagnostic and epidemiological purposes, seven MAbs were tested against 35 Brazilian rabies virus isolates from different hosts (bovines, equines, humans, bats, dogs, and foxes) on IIF assay. Based on the observation of fluorescent foci, three different reactivity patterns were observed: negative, weakly positive or positive reaction. As shown in Table 1, MAb 6H8 recognized all the virus isolates tested. LIA 02, 3E6, and 9C7 failed to recognize only one or two of the virus isolates. MAb 8B6 reacted with about half of the isolates, while 7B7 and 8D11 recognized only six and twelve isolates, respectively. Comparisons between the reactivity of the CDC MAbs panel and those MAbs described in this study are continuing, however,

previous experiments showed 8B6 recognized the seven isolates characterized as variant 2 and did not react against most of the isolates from variant 3 (Table 1). 3.3. Reactivity of MAbs against inactivated rabies virus present in commercial vaccines The reactivity of MAbs against rabies virus vaccine strains was evaluated to determine if MAbs can be used for the standardization of an enzyme-immunoassay to determine rabies vaccine strength. One reference, two human, and fourteen veterinary vaccines were used in an immunodot-blot assay, where LIA 02, 6H8, and 9C7 reacted against all vaccine strains tested. The other MAbs, except for 8D11, recognized almost 90% of the vaccine strains, and 8D11

Table 2 Monoclonal antibody (MAb) designation, protocol of fusion through which MAbs were obtained, isotype, neutralization activity, and reactivity against protein rGERA179–281. MAbs

Protocol of fusion

Isotype

Virus-neutralizing activity

Reaction against rGERA179–281

LIA02 6H8 7B7 2A5 2D2 1H2 3E6 8D11 9C7 8B6

1 2 2 3 3 4 5 5 5 5

IgG2b␬ IgG2a␬ IgM␬ IgG3␬ IgG3␬ IgG3␬ IgG3␬ IgG2a␬ IgM␬ IgG2a␬

− − − − − − − + − −

− − + − − − − − − −

−: negative; +: positive.

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Fig. 1. Immunoreactivity of monoclonal antibodies against rabies virus PV strain determined by indirect immunofluorescence in rabies-infected BHK-21 cells. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and stained with MAbs against rabies virus, followed by FITC-conjugated anti-mouse immunoglobulin. (A) LIA 02; (B) 6H8; (C) 7B7; (D) 2A5; (E) 2D2; (F) 1H2; (G) 3E6; (H) 8D11; (I) 9C7; (J) 8B6; (K) negative control anti-IMNV MAb (a non-related MAb that binds IMNV, a virus which infects marine invertebrates); (L) positive control: sera of rabies virus-immunized mice. Scale bars are 100 ␮m.

reacted against 70% of them (Table 3). The anti-IMNV MAb used as the negative control did not react with any of the vaccine strains (data not shown). 4. Discussion The present report describes the generation of ten MAbs against rabies viruses. Mice were immunized with: (1) a rabies vaccine VeroRabTM , which contains ␤-propiolactone-inactivated rabies virus, (2) three different suspensions of central nervous system tissue from mice infected with UV light-inactivated wild rabies viruses, (3) a recombinant fragment of rabies glycoprotein, rGERA179–281, and rabies virus PV strain. Six hybridomas were obtained from the fusion of mouse myeloma cells with splenocytes

from Balb/c mice immunized with rabies vaccines or with wild rabies isolates. Since these hybridomas did not produce neutralizing antibodies, the immunization of mice with the rGERA179–281 protein was performed in order to obtain the neutralizing MAbs. rGERA179–281 is a recombinant protein comprising linear epitopes (residues 179–281) from the glycoprotein of the ERA rabies virus strain expressed in Escherichia coli. By competition assay, this protein led to a measurable reduction in the ability of human rabies immunoglobulin to neutralize rabies virus (Bassi et al., 2008). The production of rabies-neutralizing antibodies in animals immunized with a fragment of the rabies glycoprotein expressed in E. coli has been described previously (Motoi et al., 2005). In the present study, immunization of mice with the recombinant protein rGERA179–281 also induced neutralizing antibodies to rabies virus,

Fig. 2. Reactivity of monoclonal antibodies against the rabies virus glycoprotein expressed by S2MtRVGP-Hy cells, determined by indirect immunofluorescence. Cells were induced to express the rabies virus glycoprotein using 700 ␮M of CuSO4 , fixed with acetone (80%), and stained with anti-rabies MAb followed by FITC-conjugated anti-mouse immunoglobulin. (A) LIA 02; (B) 8D11. The other MAbs were negative and are not shown in this figure. Scale bars are 100 ␮m.

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Table 3 Immunodot-blot assay of the immunoreactivity of monoclonal antibodies against fixed rabies virus strains employed in vaccine production. Vaccine identification

Rabies strain

LIA 02

6H8

7B7

2A5

2D2

1H2

3E6

8D11

9C7

8B6

Reference vaccine H1 H2 V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14

CVS PM Flury LEP PV PV CVS Paris Pasteur WI−38 WI−38 PV PV ERA PM CVS PV-12 PV-12 PV-12

+ + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + +

+ + − + + − + + + + + + + + + + +

+ + + + + + + + − + + + + + + + +

+ + + + + + + + − + + − + + + + +

+ + + + + + + + − + + − + + + + +

+ + + + + + + + − + + + + + + + +

− + + + + − + + − + − + − + + + +

+ + + + + + + + + + + + + + + + +

+ + + + + + + + − + + + + + + + +

−: negative; +: positive; H1 and H2: human vaccines; V1–V14: veterinary vaccines.

although in low titers (data not shown). Four MAbs were obtained from this fusion, including a rabies neutralizing MAb. However, surprisingly, a strong immune response also was induced by the boost in mice not immunized previously with the rGERA179–281 protein (data not shown), and none of these four MAbs recognized the recombinant protein. Together, these data suggest the boost with rabies virus PV strain (not the rGERA179–281 protein) was responsible for the production of MAbs 3E6, 9C7, 8D11, and 8B6. In the present work, a relatively limited amount of useful MAbs was obtained for each immunization protocol. In fact, on average <1–5% of the growth-positive wells during a typical fusion will contain hybridomas that secrete the desired antibody (Yokoyama et al., 2006). However, the number of hybridomas achieved in the fusion process can vary widely according to different authors. For example, Benmansour et al. (1991) immunized mice with ␤-propiolactone-inactivated CVS and obtained several hundred MAbs against rabies. On the other hand, Jiang et al. (2010) immunized mice with a sucrose-gradient-purified and ␤propiolactone-inactivated rabies virus L16 strain and obtained 25 MAbs, while Akacem et al. (1992) obtained only seven MAbs when mice were immunized with a wild rabies virus. Several possible explanations for the limited amount of MAbs obtained in the present report may exist. One explanation could be the great instability of hybrid cells in the first days after fusion due to the loss of some chromosomes (Köhler and Milstein, 1975), which may result in non-antibody-producing hybridomas or in hybrid cells that are unsuitable for survival in HAT culture medium (Taggart and Samloff, 1983; Zola, 1987). Non-secreting hybridomas and clones with an unstable assortment of chromosomes may also be present in the original well and may outgrow and replace the desired antibody-producing hybridomas (Sheehan, 2007). The use of non-purified antigens in the immunization process, such as rabies vaccine or infected mouse brain suspension, may result in the production of MAbs against many other antigens (Yokoyama et al., 2006). In the present work, many hybridomas with a positive result in the screening test secreted antibodies that cross-reacted with non-infected BHK-21 cells (data not shown), possibly due to the presence of the non-purified antigens. Seven MAbs described in this study showed different reactivity against wild rabies virus isolates on immunofluorescence assays, with MAbs recognizing varying numbers of isolates from six to all of the 35 isolates tested. Among them, LIA 02, 6H8, 3E6, and 9C7 recognized all or almost all of the isolates tested, suggesting that these MAbs may be important in the future as diagnostic tools. For example, these MAbs may be labeled with FITC and used for routine diagnosis.

Currently, the most widely used test for the detection of rabies antigens is the direct fluorescent antibody test, which uses FITC-conjugated anti-rabies antibodies (Woldehiwet, 2005). These conjugated antibodies often are obtained from the sera of immunized animals, which may contain non-rabies antibodies (Dean et al., 1996). The use of MAbs may increase the specificity of the conjugate, reduce the variation between different batches, and also help to distinguish rabies virus from other types of Lyssavirus. The MAbs generated in the present study also may be useful for the development of rapid immunoassays such as ELISA, latex agglutination test, and immunochromatographic strip test to detect rabies virus in infected animals (Kang et al., 2007; Kasempimolporn et al., 2000; Nishizono et al., 2008) or to quantify anti-rabies serum neutralizing antibodies in immunized animals (Shiota et al., 2009; Wang et al., 2010; Zhang et al., 2009). The use of MAb panels also allowed the identification of common rabies virus variants and the enhancement of epidemiological studies, serving as a useful tool for geographical distribution analysis of the virus and animals hosts around the world, including the USA (Crawford-Miksza et al., 1999), Europe, and Russia (Bourhy et al., 1992; Metlin et al., 2004). Studies with isolates from Latin America are developed mainly using a panel of eight MAbs provided by CDC (Castilho et al., 2010; Diaz et al., 1994). By identifying the rabies virus antigenic pattern, these studies permit the identification of the most likely rabies transmitter or rabies reservoir. However, as demonstrated by Favoretto et al. (2002), antigenic variants have been identified in Brazil which are not compatible with the current panel of MAbs used to characterize isolates in the Americas. In turn, this identification of new antigenic profiles restricts the use of the current MAb panel to identify potentially emergent rabies reservoirs, suggesting new MAbs are needed to complement this panel. Comparisons between the reactivity of the seven MAbs described here against wild rabies isolates and the panel of MAbs provided by CDC showed that 8B6 is the most promising MAb for this purpose. In this present report, 8B6 reacted against all seven isolates characterized as variant 2, and did not react against most of the isolates from variant 3, suggesting 8B6 could be employed in epidemiological studies. However, further studies with a greater number of isolates still are needed in order to verify if the reactivity of 8B6 against isolates from different variants is compatible with the genetic characterization of the isolates, including variants which are incompatible with the profiles defined in the panel of MAbs provided by CDC. Vaccine strength is currently determined by the NIH test, which uses a large number of live animals and presents several problems in terms of reproducibility (Barth et al., 1988; Wilbur and

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Aubert, 1996). A new assay to replace the NIH test may not only overcome these problems, but, as an advantage, would also avoid handling live rabies virus. MAbs, especially those specific against the rabies glycoprotein, have been employed in the development of immunoassays, such as ELISA, to determine vaccine strength and for quality control during vaccine production (Fournier-Caruana et al., 2003; Nagarajan et al., 2006). These methods are based on determining the antigen content of vaccine preparations, i.e., measuring the content of glycoprotein and/or RNP. In the present study, the reactivity of the MAbs against fixed rabies virus strains present in commercial vaccines was evaluated. One reference, two human, and fourteen veterinary vaccines, comprising eight different rabies strains, were tested. Three MAbs reacted against rabies virus strains present in all the vaccines tested and the remaining MAbs recognized twelve or more vaccine strains, as demonstrated by immunodot-blot assay. These results warrant further studies on the use of MAbs for the standardization of an immunoassay to determine the strength of rabies vaccines. The results presented in this paper suggest the most promising MAbs to be employed in further studies are LIA 02, 6H8, 3E6, and 9C7, which recognized more than 94% of wild rabies isolates, and 8D11, which showed neutralizing activity against rabies virus. The anti-glycoprotein specificity of MAb 8D11, which presents an IIF staining pattern located predominantly on the cell membrane, was confirmed by IIF on S2MtRVGP-Hy cells. This MAb also showed rabies virus-neutralizing activity in vitro, preventing the infection of BHK-21 cells by rabies virus. These results support the investigation of the protective activity of MAb 8D11 in vivo. The in vivo protective activity of some MAb cocktails has been investigated by other authors as a possible post-exposure treatment for rabies (Bakker et al., 2008; Müller et al., 2009). In conclusion, the present report describes the generation of ten MAbs against rabies virus. The reactivity pattern of MAb 8B6 with wild rabies virus strains, when compared with the panel of MAbs provided by CDC, highlights the potential of 8B6 to be employed in epidemiological studies. In addition, MAb 8D11 was able to neutralize the virus in vitro, and MAbs 6H8, LIA 02, 3E6, and 9C7 recognized a greater number of wild rabies virus isolates and fixed rabies strains present in the commercial vaccines tested. Therefore, these MAbs may be considered the most promising to be employed in further diagnosis and immunoassay studies. Acknowledgments The authors thank Dr. Wlamir Moura (Instituto Nacional de Controle de Qualidade em Saúde – INCQS, Rio de Janeiro, RJ), DVM Fernando José Pires de Souza and Amiris Pereira Gonc¸alves de Campos (Laboratório Nacional Agropecuário do Ministério da Agricultura – LANAGRO, Campinas, SP) for kindly providing the vaccine samples. The authors are also grateful to Dr. Carlos Augusto Pereira and Dr. Renato Mancini Astray (Instituto Butantan, São Paulo, SP) for graciously providing the S2MtRVGP-Hy cells. This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Ministério da Agricultura, Pecuária e Abastecimento (MAPA), Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), and Programa Institucional de Bolsas de Iniciac¸ão Científica (PIBIC/UFSC). References Akacem, O., Benmansour, A., Coulon, P., Brahimi, M., Benhassine, M., 1992. Production of monoclonal antibodies against a wild strain of rabies virus. Archives de l’Institut Pasteur d’Algérie 58, 283–290. Bakker, A.B.H., Python, C., Kissling, C.J., Pandya, P., Marissen, W.E., Brink, M.F., Lagerwerf, F., Worst, S., van Corven, E., Kostense, S., Hartmann, K., Weverling, G.J., Uytdehaag, F., Herzog, C., Briggs, D.J., Rupprecht, C.E., Grimaldi, R., Goudsmit, J., 2008. First administration to humans of a monoclonal antibody cocktail

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