Original Paper
Intervirology 1992;33:17-22
André D. M bidaa Odette G. Gaudina Odile Sabidob Bruno Pozzettoa Jean-Claude Le Bihana-1
Monoclonal Antibody Specific for the Cellular Receptor of Echoviruses
Department o f Virology and Fluocytometry Unit, Faculty o f Medicine, CHU, St-Etienne, France
Summary Cell lines of primate origin carry membrane receptors which are specific for echoviruses (EV). The present report describes isola tion and characterization of a monoclonal antibody (Mab 143) reacting with the membrane of KB cells. The Mab was selected for its protection of different cell lines from primate origin against the CPE of EV-11. This protection was found to extend to most EV serotypes and to coxsackievirus A9, while the replica tion of several other picornaviruses was not affected. The fluorecein isothiocyanate labelled Mab did not react with cell lines from bovine, canine, or rabbit origin, but bound specifically to the cell lines from human or simian origin. These results suggest that a unique receptor site is used by most EV serotypes for binding onto and penetration into susceptible cells.
Introduction The echoviruses (EVs) represent a subgroup of the picornavirus family and contain 31 antigenically distinct serotypes [1]. These viruses are responsible for many symptoms in humans [2, 3], and, like other enteroviruses, they attach to a specific receptor site localized on the surface of the susceptible cell membrane [4],
1 We are grateful to Mrs. M.H. Richard for technical support during the preparation of hybridomas.
Received: July 1,1990 Accepted: January 24,1991
Previous reports have demonstrated that the different groups of picornaviruses absorb to distinct receptors for attachment onto and en try into cells [5]. Competition binding studies have established the existence of several recep tor families: one for polioviruses, two for rhinoviruses, one for foot-and-mouth diseases vi ruses, one for encephalomyocarditis viruses, and one for coxsackievirus group B [5-8].
Prof. Odette G. Gaudin Department of Virology Faculty of Medicine, 15, rue Ambroise-Parc F-42023 St-Etienne Cedex (France)
€>1992 S. Karger AG, Basel 0300-5526/92/ 0331-001752.75/0
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/24/2018 3:58:11 PM
Key Words Cellular receptor Echovirus Coxsackievirus Picornavirus Monoclonal antibody Cytofluorimetry
Material and Methods Cells Human KB cell line (derived from a nasopharynx carcinoma) and P2002 strain (embryonic lung fibro blasts; Flow Laboratories) were maintained in MEM supplemented with 10% fetal calf serum, 2 m M E-gluta mine, 100 lU /m l penicillin, and 100 pg/m l streptomy cin. The Wish cell line (derived from a human amnios) was cultured in the same medium but 1% glucose was added instead of /^glutamine. Vero and BGM cell lines (from simian origin) were cultured in E 199 medium supplemented as above. Non-primate cells, including rabbit kidney (RK13), Madin Darby bovine kidney (MDBK), and Madin Darby canine kidney (MDC'K) lines, were maintained in the medium described for Wish cells. Viruses For EVs and coxsackieviruses both prototype strains and clinical isolates serotyped with specific an tisera (Statens Seruminstitut, Copenhagen, Denmark) were used. Polioviruses were attenuated vaccinal Sabin strains (Institut Merieux, France). Human rhinovirus 14 (a gift from M. Aymard and J.J. Chomel, Lyon, France) was a clinical isolate. EV-ll stock solution was produced in large amounts on KB cells, concentrated by high-speed centrifugation for 3 h at 180,000 gat 4° and purified on CsS04gradient
[51For some experiments, the purified virus was biotin ylated as previously described [14,15], using a commer cial kit (Amersham, France). The biotinylated virus was aliquoted and stored at -80° until used. Cell Protection Assay This assay was used for studying the antireceptor activity of the Mab. Cells (30,000/well) were seeded in 96-well microplates and incubated overnight at 37°. The cells were then pretreated with 50 pi of a concentration of 1 mg/ml of unpurified ascites for 1 h at 37°. (This antibody concentration was optimized by serial titration
18
in preliminary experiments.) The antibody was re moved, and 100 pi of virus at dilutions ranging from 10 ' to 10 6 of stock solution was added to yield CPE within 48 h. Four wells per dilution were tested both in the presence and absence of antibody for each viral dilu tion. The results were expressed as logm reduction in titer at the concentration of 20 pg of unpurified ascites per well. Production o f Receptor-Specific Mab KB cell membrane extracts were treated with deter gents and semipurified by high-pressure liquid chroma tography, (HPLC), as described elsewhere [Mbida et al., submitted]. The HPLC fraction used for the production of the Mab was chosen for its ability to bind EV-ll and consisted in a peak showing a molecular weight of 220,000-240,000. Adult OF1 Swiss mice (IFFA-C'redo: France) were immunized intraperitoneally at monthly intervals with 40 pg of the HPLC fraction described above, diluted 1:2 in complete Freund's adjuvant for the first injection and diluted 1:2 in incomplete Freund's adjuvant for the second and third injections. The mice were bled before each injection, and sera were ex amined for evidence of antibodies protecting KB cells against EV-ll infection. The positive mice were given a final boost without Freund’s adjuvant 3 days before the cell fusion. Mouse spleen cells were collected and fused with SP2/0 cells [10]. Supernatants from growing hybridomas were assayed in the cell protection test 10-20 days after the cell fusion. The positive hybridomas were subcloned at least three times by the limit dilution method [14], The resulting clones were controlled for the production of efficient antibodies in the cell protection test and assayed for determination of the isotype sub class using a commercial kit (Amersham, France). In cytofluorometry experiments, the antibody con tained in culture fluids was concentrated by (NHj) 2SOj precipitation, resuspended in phosphate-buffered sa line (PBS), dialyzed overnight at 4° against PBS, and labelled with fluorescein isothiocyanate (FITC), as pre viously described [14]. For the cell protection assay with different picornaviruses, ascites tumors were produced by injecting intra peritoneally 10 million cells per mouse to Balb/c mice pretreated with 0.5 ml of pristane and immunosuppressed with cortisone acetate. Flow Cytometric Analysis For binding studies w'ith EV-ll, 500,000 cells in a Becton test tube were incubated with biotinylated EV-ll for 30 min at 4°; FITC-labelled streptavidin (Amers ham) diluted 1:25 in PBS was then added and allowed to incubate at 4° for 30 min. The cells were washed and
Mbida/Gaudin/Sabido/Pozzetto/ Le Bihan
Monoclonal Antibody to Echovirus Cell Receptor
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/24/2018 3:58:11 PM
Monoclonal antibodies (Mabs) specific for the cellular receptor sites of these virus groups have been isolated [7,9-13]. We now report the production of a mouse Mab which is effective in specifically blocking attachment and in fection capacity of several EV serotypes.
kept in PBS-1% formaldehyde at 4° until used for cytofluorometric analysis. For binding studies with the antireceptor Mab puri fied from cell culture supernatant, 500,000 cells per tube were incubated for 50 min at 4° with the appropriate predetermined concentration of FITC-labelled Mab previously absorbed with calf liver powder. Cell wash ings and fixation were performed as described above. In some experiments kinetic binding studies were performed with different incubation times between EVII or Mab and cells. Flow cytometric analysis was performed using a Facstar Plus cell sorter (Becton Dickinson, Montain View, Calif., USA) equipped with a 5.0-watt argon laser (Innova 90: Coherent. Palo Alto, Calif.), emitting 250 m W at 488 nm. Data were processed on a Hewlett- Pack ard computer system (HP 300-900), using the Facstar research program. Green fluorescence signals were col lected through a bandpass filter (530/30 nm). All signals were input in internal processing, using 1,024 channels. Debris and dead cells were excluded from analysis with a gate built according to forward light scatter and per pendicular light scatter. Cytogram fluorescent para meters were recorded after linear amplification. Statis tics were calculated on 10,000 cells. The threshold of positivity for green fluorescence was arbitrarily estab lished on the basis of the negative control sample of cells. One-color analysis data were depicted by histo grams in which the number of cells appeared on the ordinate and the intensity of fluorescence on the abscis sa [16]. The rate constants of association between cells and EV-U or Mab were calculated as described by Rueckert [171-
Results
Table 1. Ability of Mab 143 to prevent P2002 cells from infection with different EVs
EV serotype
1 2 3 4 5 6 7 9 II 12 13 14 15 16 17 18 19 20 21 24 25 26 27 29 30 31 32 33
Log10 reduction in titer“ prototype strains
clinical isolates
1.7 2.9 NT NT NT NT NT NT 2.5 NT 2.9 NT 1.7 NT 2.3 1.7 1.7 2.3 NT NT NT 2.1 2.9 2.5 2.3 NT 2.3 NT
NT NT 2.5 1.7 2.3 2.3 2.3 2.3 2.5 2.5 NT 2.3 NT 1.7 1.7 1.7 NT 1.7 2.3 2.3 2.3 NT NT 1.7 1.7 1.7 NT 2.5
N T = N o t tested. “ Figures represent the reduction of the virus titer (titer of the virus on untreated cells - titer of the virus on cells treated with Mab 143).
Selection and Characterization o f the Antireceptor Mab
Viral and Cellular Specifities o f Mab 143 Specificity o f M ab 143fo r the Receptor o fE V
KB, P2002, Vero, or Wish cells were pretreated with ascitic Mab 143 and then challenged with different EVs. As shown in table 1, all the EV serotypes tested were inhibited, with the logic reduction in titer ranging from 1.7 to 2.9. Sever-
19
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/24/2018 3:58:11 PM
Of 1,100 culture supernatant wells, 3 were found positive and subcloned. Two positive hybridomas were lost during subcloning exper iments; the last hybridoma, called 143, yielded four clones: 143/3/13, 143/3/116, 143/3/J4, and 143/3/J13. All Mabs produced from these clones belonged to the murine isotype Gl, with kappa light chain; they will be referenced as Mab 143.
Table 2. Ability of Mab 143 to prevent P2002 or KB cells from infection with picomaviruses other than EVs
Picomavirusesa
Logio reduction in titer*5
Cells used in protection assay
Polio 1 Polio 2 Polio 3 Coxsackie A9 Coxsackie A16 Coxsackie A2I Coxsackie Bl Coxsackie B2 Coxsackie B3 Coxsackie B4 Coxsackie B5 Coxsackie B6 Human rhinovirus 14
Intervirology 1992;33:17-22
André D. M bidaa Odette G. Gaudina Odile Sabidob Bruno Pozzettoa Jean-Claude Le Bihana-1
Monoclonal Antibody Specific for the Cellular Receptor of Echoviruses
Department o f Virology and Fluocytometry Unit, Faculty o f Medicine, CHU, St-Etienne, France
Summary Cell lines of primate origin carry membrane receptors which are specific for echoviruses (EV). The present report describes isola tion and characterization of a monoclonal antibody (Mab 143) reacting with the membrane of KB cells. The Mab was selected for its protection of different cell lines from primate origin against the CPE of EV-11. This protection was found to extend to most EV serotypes and to coxsackievirus A9, while the replica tion of several other picornaviruses was not affected. The fluorecein isothiocyanate labelled Mab did not react with cell lines from bovine, canine, or rabbit origin, but bound specifically to the cell lines from human or simian origin. These results suggest that a unique receptor site is used by most EV serotypes for binding onto and penetration into susceptible cells.
Introduction The echoviruses (EVs) represent a subgroup of the picornavirus family and contain 31 antigenically distinct serotypes [1]. These viruses are responsible for many symptoms in humans [2, 3], and, like other enteroviruses, they attach to a specific receptor site localized on the surface of the susceptible cell membrane [4],
1 We are grateful to Mrs. M.H. Richard for technical support during the preparation of hybridomas.
Received: July 1,1990 Accepted: January 24,1991
Previous reports have demonstrated that the different groups of picornaviruses absorb to distinct receptors for attachment onto and en try into cells [5]. Competition binding studies have established the existence of several recep tor families: one for polioviruses, two for rhinoviruses, one for foot-and-mouth diseases vi ruses, one for encephalomyocarditis viruses, and one for coxsackievirus group B [5-8].
Prof. Odette G. Gaudin Department of Virology Faculty of Medicine, 15, rue Ambroise-Parc F-42023 St-Etienne Cedex (France)
€>1992 S. Karger AG, Basel 0300-5526/92/ 0331-001752.75/0
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/24/2018 3:58:11 PM
Key Words Cellular receptor Echovirus Coxsackievirus Picornavirus Monoclonal antibody Cytofluorimetry
Material and Methods Cells Human KB cell line (derived from a nasopharynx carcinoma) and P2002 strain (embryonic lung fibro blasts; Flow Laboratories) were maintained in MEM supplemented with 10% fetal calf serum, 2 m M E-gluta mine, 100 lU /m l penicillin, and 100 pg/m l streptomy cin. The Wish cell line (derived from a human amnios) was cultured in the same medium but 1% glucose was added instead of /^glutamine. Vero and BGM cell lines (from simian origin) were cultured in E 199 medium supplemented as above. Non-primate cells, including rabbit kidney (RK13), Madin Darby bovine kidney (MDBK), and Madin Darby canine kidney (MDC'K) lines, were maintained in the medium described for Wish cells. Viruses For EVs and coxsackieviruses both prototype strains and clinical isolates serotyped with specific an tisera (Statens Seruminstitut, Copenhagen, Denmark) were used. Polioviruses were attenuated vaccinal Sabin strains (Institut Merieux, France). Human rhinovirus 14 (a gift from M. Aymard and J.J. Chomel, Lyon, France) was a clinical isolate. EV-ll stock solution was produced in large amounts on KB cells, concentrated by high-speed centrifugation for 3 h at 180,000 gat 4° and purified on CsS04gradient
[51For some experiments, the purified virus was biotin ylated as previously described [14,15], using a commer cial kit (Amersham, France). The biotinylated virus was aliquoted and stored at -80° until used. Cell Protection Assay This assay was used for studying the antireceptor activity of the Mab. Cells (30,000/well) were seeded in 96-well microplates and incubated overnight at 37°. The cells were then pretreated with 50 pi of a concentration of 1 mg/ml of unpurified ascites for 1 h at 37°. (This antibody concentration was optimized by serial titration
18
in preliminary experiments.) The antibody was re moved, and 100 pi of virus at dilutions ranging from 10 ' to 10 6 of stock solution was added to yield CPE within 48 h. Four wells per dilution were tested both in the presence and absence of antibody for each viral dilu tion. The results were expressed as logm reduction in titer at the concentration of 20 pg of unpurified ascites per well. Production o f Receptor-Specific Mab KB cell membrane extracts were treated with deter gents and semipurified by high-pressure liquid chroma tography, (HPLC), as described elsewhere [Mbida et al., submitted]. The HPLC fraction used for the production of the Mab was chosen for its ability to bind EV-ll and consisted in a peak showing a molecular weight of 220,000-240,000. Adult OF1 Swiss mice (IFFA-C'redo: France) were immunized intraperitoneally at monthly intervals with 40 pg of the HPLC fraction described above, diluted 1:2 in complete Freund's adjuvant for the first injection and diluted 1:2 in incomplete Freund's adjuvant for the second and third injections. The mice were bled before each injection, and sera were ex amined for evidence of antibodies protecting KB cells against EV-ll infection. The positive mice were given a final boost without Freund’s adjuvant 3 days before the cell fusion. Mouse spleen cells were collected and fused with SP2/0 cells [10]. Supernatants from growing hybridomas were assayed in the cell protection test 10-20 days after the cell fusion. The positive hybridomas were subcloned at least three times by the limit dilution method [14], The resulting clones were controlled for the production of efficient antibodies in the cell protection test and assayed for determination of the isotype sub class using a commercial kit (Amersham, France). In cytofluorometry experiments, the antibody con tained in culture fluids was concentrated by (NHj) 2SOj precipitation, resuspended in phosphate-buffered sa line (PBS), dialyzed overnight at 4° against PBS, and labelled with fluorescein isothiocyanate (FITC), as pre viously described [14]. For the cell protection assay with different picornaviruses, ascites tumors were produced by injecting intra peritoneally 10 million cells per mouse to Balb/c mice pretreated with 0.5 ml of pristane and immunosuppressed with cortisone acetate. Flow Cytometric Analysis For binding studies w'ith EV-ll, 500,000 cells in a Becton test tube were incubated with biotinylated EV-ll for 30 min at 4°; FITC-labelled streptavidin (Amers ham) diluted 1:25 in PBS was then added and allowed to incubate at 4° for 30 min. The cells were washed and
Mbida/Gaudin/Sabido/Pozzetto/ Le Bihan
Monoclonal Antibody to Echovirus Cell Receptor
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/24/2018 3:58:11 PM
Monoclonal antibodies (Mabs) specific for the cellular receptor sites of these virus groups have been isolated [7,9-13]. We now report the production of a mouse Mab which is effective in specifically blocking attachment and in fection capacity of several EV serotypes.
kept in PBS-1% formaldehyde at 4° until used for cytofluorometric analysis. For binding studies with the antireceptor Mab puri fied from cell culture supernatant, 500,000 cells per tube were incubated for 50 min at 4° with the appropriate predetermined concentration of FITC-labelled Mab previously absorbed with calf liver powder. Cell wash ings and fixation were performed as described above. In some experiments kinetic binding studies were performed with different incubation times between EVII or Mab and cells. Flow cytometric analysis was performed using a Facstar Plus cell sorter (Becton Dickinson, Montain View, Calif., USA) equipped with a 5.0-watt argon laser (Innova 90: Coherent. Palo Alto, Calif.), emitting 250 m W at 488 nm. Data were processed on a Hewlett- Pack ard computer system (HP 300-900), using the Facstar research program. Green fluorescence signals were col lected through a bandpass filter (530/30 nm). All signals were input in internal processing, using 1,024 channels. Debris and dead cells were excluded from analysis with a gate built according to forward light scatter and per pendicular light scatter. Cytogram fluorescent para meters were recorded after linear amplification. Statis tics were calculated on 10,000 cells. The threshold of positivity for green fluorescence was arbitrarily estab lished on the basis of the negative control sample of cells. One-color analysis data were depicted by histo grams in which the number of cells appeared on the ordinate and the intensity of fluorescence on the abscis sa [16]. The rate constants of association between cells and EV-U or Mab were calculated as described by Rueckert [171-
Results
Table 1. Ability of Mab 143 to prevent P2002 cells from infection with different EVs
EV serotype
1 2 3 4 5 6 7 9 II 12 13 14 15 16 17 18 19 20 21 24 25 26 27 29 30 31 32 33
Log10 reduction in titer“ prototype strains
clinical isolates
1.7 2.9 NT NT NT NT NT NT 2.5 NT 2.9 NT 1.7 NT 2.3 1.7 1.7 2.3 NT NT NT 2.1 2.9 2.5 2.3 NT 2.3 NT
NT NT 2.5 1.7 2.3 2.3 2.3 2.3 2.5 2.5 NT 2.3 NT 1.7 1.7 1.7 NT 1.7 2.3 2.3 2.3 NT NT 1.7 1.7 1.7 NT 2.5
N T = N o t tested. “ Figures represent the reduction of the virus titer (titer of the virus on untreated cells - titer of the virus on cells treated with Mab 143).
Selection and Characterization o f the Antireceptor Mab
Viral and Cellular Specifities o f Mab 143 Specificity o f M ab 143fo r the Receptor o fE V
KB, P2002, Vero, or Wish cells were pretreated with ascitic Mab 143 and then challenged with different EVs. As shown in table 1, all the EV serotypes tested were inhibited, with the logic reduction in titer ranging from 1.7 to 2.9. Sever-
19
Downloaded by: Leiden University Medisch Centrum 132.229.13.63 - 10/24/2018 3:58:11 PM
Of 1,100 culture supernatant wells, 3 were found positive and subcloned. Two positive hybridomas were lost during subcloning exper iments; the last hybridoma, called 143, yielded four clones: 143/3/13, 143/3/116, 143/3/J4, and 143/3/J13. All Mabs produced from these clones belonged to the murine isotype Gl, with kappa light chain; they will be referenced as Mab 143.
Table 2. Ability of Mab 143 to prevent P2002 or KB cells from infection with picomaviruses other than EVs
Picomavirusesa
Logio reduction in titer*5
Cells used in protection assay
Polio 1 Polio 2 Polio 3 Coxsackie A9 Coxsackie A16 Coxsackie A2I Coxsackie Bl Coxsackie B2 Coxsackie B3 Coxsackie B4 Coxsackie B5 Coxsackie B6 Human rhinovirus 14
