Original Paper
Intervirology 1992;86:86-93
Ranajit Pal Fulvia di Marzo Veronese B. C. Nair Rukhsana Rahman George Hoke Steven W. Mumbauer M. G. Samgadharan
Characterization of a Neutralizing Monoclonal Antibody to the External Glycoprotein of HIV-1
Advanced BioScience Laboratories, Kensington, Md., USA
Introduction The envelope gene of HIV-1 is organized with gpl60 as the primary translational product and gpl20 and gp41 as the external and trans-
Received : November 7,1989 Accepted : June 26,1992
membrane glycoproteins, respectively [1, 2], Some of the major efforts in designing an antiHIV-1 vaccine have been directed towards studies of these envelope glycoproteins, since these proteins contain epitopes naturally ac-
Ranajit Pal Advanced BioScience Laboratories, Inc. Kensington. MD 20895 (USA)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 9:49:50 PM
Key Words Human immunodeficiency virus type 1 Neutralizing epitope External glycoprotein Syncytia formation Monoclonal antibody
Summary The major neutralizing epitope on the external glycoprotein of HIV-1 was studied with an envelope-specific monoclonal anti body and with a human serum positive for antibodies to HIV-1 proteins, both of which were able to neutralize virus infectivity. The monoclonal antibody reacted specifically with gpl20 from HI V-1]hb, and was shown to neutralize infection of CEM cells by cell-free virions, and inhibited the formation of syncytia nor mally observed when uninfected cells are cocultured with HIV1-infected cells. Similar neutralization of viral infection and inhibition of syncytia formation was also demonstrated by the HIV-l-antibody-positive human serum. By examining a number of overlapping peptides from a region of HIV-1 gpl20 known to contain a neutralizing epitope, this epitope was localized be tween amino acids 307 and 320 (V3 loop) in the external glyco protein molecule. The monoclonal antibody did not interfere with the binding of gpl20 to CD4, or with the subsequent step of CD4-induced shedding of gpl20 from the viral envelope. How ever, it blocked the proteolytic cleavage of the V3 loop by thrombin, suggesting that the antibody may be inhibiting the interaction of the loop with other membrane-bound proteins.
Materials and Methods Neutralization Assay o f Cell-Free Virus The HIV-l-positive serum was heat inactivated for 30 min at 57° and subjected to serial 2-fold dilution in complete RPMI-1640 medium. IgGs from the mono clonal antibody were affinity purified and similarly serially diluted in complete RPMI-1640 medium. A 100-.ul aliquot of each dilution of the respective antibody was incubated with HlV-lmB virus stock containing 25.000 cpm reverse-transcriptase activity for 60 min at 4° and then for 15 min at room temperature. CEM cells (1 x 105) were then added to the virus-antibody mixture and incubated for 60 min at 37°. The cells were sup plemented with 2 ml of complete RPM1 medium and transferred to 6-well plates. Two millilitres o f RPMI medium were added after 24 h and the reverse-tran scriptase activity of the supernatant was determined on day 7 after infection. Syncytium Inhibition Assay The syncytia assay was performed in 96-well plates by coculturing I x I05 CEM cells with 5 x 103 M olt3/ HlV-lnm cells (Molt 3 cells chronically infected with HTLV-II1B isolate) as described elsewhere [15]. AntiHIV-1-positive human serum or the monoclonal anti body were present in the medium throughout the culture period. Immunoprécipitation Assay Molt3/HIV-l,||B cells were labeled with [35S]methionine and immunoprecipitated by the monoclonal an tibody and by H IV-l-antibody-positive human serum as described elsewhere [16]. Monoclonal Antibody Preparation BALB/c mice (Charles River Breeding Labora tories), were immunized by successive intraperitoneal inoculations of 20 pg purified gpl20 from HTLV-1IIB emulsified in complete Freund’s adjuvant for the first inoculation and in incomplete Freund's adjuvant for the following five boosters (10 ug of protein each time), given 1 week apart. Three days after a final intraperito neal booster with 20 gg gpl20 in phosphate-buffered saline, splenic lymphocytes were fused with the NS-1 mouse myeloma cell line. The fusion procedure, cell culturing, determination of immunoglobulin subclass, and cloning of hybridoma lines were very similar to procedures previously described in the literature [17]. Mouse ascitic fluid containing monoclonal antibodies was prepared as described previously [17],
87
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cessible to the immune system. Antibodies ca pable of neutralizing HIV-I infection of target cells are usually found in serum from HI V-l-infected individuals, albeit in low concentrations relative to those induced by other viruses [3-5]. Most of these antibodies bind gpl20 and neu tralize more than one serologically distinct iso late of the virus [5], In addition, native gpl20 and recombinant gpl60 and gpl20 have been shown to elicit type-specific antibodies that neutralize the infectivity of HIV-1 in vitro [5,6]. The major neutralizing epitope has been nar rowed down to a 24-amino-acid segment from amino acids 302 to 325 (V3 loop) [5,6]. Murine monoclonal antibodies directed towards this region blocked syncytia formation and neu tralized infectivity of HIV-1 [reviewed in ref. 5,6]. The major pathway of infection of T cells by HIV-1 involves pH-independent fusion of viral and host cell membranes [7, 8], This process is initiated by the interaction of gpl20 with its receptor, CD4 [9-11]. After gpl20 has bound to CD4, a conformational change is induced on the glycoprotein molecule which enhances the susceptibility of the V3 loop to protease diges tion, and eventually dissociates gpl20 from the viral membrane [12]. This results in the expo sure of the amino terminal hydrophobic do main of gp41 which initiates the fusion process [13,14]. In this communication, an HIV-1-antibodypositive human serum and a monoclonal anti body directed towards the external envelope glycoprotein have been used to further define the neutralizing epitope in the gpl20 molecule. Moreover, the monoclonal antibody was shown to inhibit a post-binding event during HIV-1 infection, indicated by its blocking the cleavage of the V3 loop by an exogenous pro tease.
Results
88
Fig. 1. Reactivity of M77 and PS12 with HIV-l pro teins. Molt3/HIV-lmB cells were labeled with [,5S]methionine for 7 h and the clarified lysate was immunoprecipitated with M77 (A, lane I) and PS12 (B. lane I) as described in Materials and Methods. For tunicamycin treatment the cells were pretreated for I h, labeled for 7 h in the presence of 1.5 pg/ml of tunicamycin and immunoprecipitated with M77 (A, lane 2) and PSI2 (B, lane 2).
of gpl20, as it was found to react with the deglycosylated 80-kD protein generated in the infected cell by tunicamycin treatment (fig. IB, lane 2). Biological Activity o f Monoclonal Antibody M77 and Human Serum PS12 Purified IgG from M77 was tested for syncy tia-blocking activity and for virus neutraliza tion. For the syncytia-blocking assay, the OEM cells were cocultured with Molt3/HIV-ImB cells in the presence of the antibody. Nearly 90% of the inhibition of syncytia was achieved at a concentration
Intervirology 1992;86:86-93
Ranajit Pal Fulvia di Marzo Veronese B. C. Nair Rukhsana Rahman George Hoke Steven W. Mumbauer M. G. Samgadharan
Characterization of a Neutralizing Monoclonal Antibody to the External Glycoprotein of HIV-1
Advanced BioScience Laboratories, Kensington, Md., USA
Introduction The envelope gene of HIV-1 is organized with gpl60 as the primary translational product and gpl20 and gp41 as the external and trans-
Received : November 7,1989 Accepted : June 26,1992
membrane glycoproteins, respectively [1, 2], Some of the major efforts in designing an antiHIV-1 vaccine have been directed towards studies of these envelope glycoproteins, since these proteins contain epitopes naturally ac-
Ranajit Pal Advanced BioScience Laboratories, Inc. Kensington. MD 20895 (USA)
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 9:49:50 PM
Key Words Human immunodeficiency virus type 1 Neutralizing epitope External glycoprotein Syncytia formation Monoclonal antibody
Summary The major neutralizing epitope on the external glycoprotein of HIV-1 was studied with an envelope-specific monoclonal anti body and with a human serum positive for antibodies to HIV-1 proteins, both of which were able to neutralize virus infectivity. The monoclonal antibody reacted specifically with gpl20 from HI V-1]hb, and was shown to neutralize infection of CEM cells by cell-free virions, and inhibited the formation of syncytia nor mally observed when uninfected cells are cocultured with HIV1-infected cells. Similar neutralization of viral infection and inhibition of syncytia formation was also demonstrated by the HIV-l-antibody-positive human serum. By examining a number of overlapping peptides from a region of HIV-1 gpl20 known to contain a neutralizing epitope, this epitope was localized be tween amino acids 307 and 320 (V3 loop) in the external glyco protein molecule. The monoclonal antibody did not interfere with the binding of gpl20 to CD4, or with the subsequent step of CD4-induced shedding of gpl20 from the viral envelope. How ever, it blocked the proteolytic cleavage of the V3 loop by thrombin, suggesting that the antibody may be inhibiting the interaction of the loop with other membrane-bound proteins.
Materials and Methods Neutralization Assay o f Cell-Free Virus The HIV-l-positive serum was heat inactivated for 30 min at 57° and subjected to serial 2-fold dilution in complete RPMI-1640 medium. IgGs from the mono clonal antibody were affinity purified and similarly serially diluted in complete RPMI-1640 medium. A 100-.ul aliquot of each dilution of the respective antibody was incubated with HlV-lmB virus stock containing 25.000 cpm reverse-transcriptase activity for 60 min at 4° and then for 15 min at room temperature. CEM cells (1 x 105) were then added to the virus-antibody mixture and incubated for 60 min at 37°. The cells were sup plemented with 2 ml of complete RPM1 medium and transferred to 6-well plates. Two millilitres o f RPMI medium were added after 24 h and the reverse-tran scriptase activity of the supernatant was determined on day 7 after infection. Syncytium Inhibition Assay The syncytia assay was performed in 96-well plates by coculturing I x I05 CEM cells with 5 x 103 M olt3/ HlV-lnm cells (Molt 3 cells chronically infected with HTLV-II1B isolate) as described elsewhere [15]. AntiHIV-1-positive human serum or the monoclonal anti body were present in the medium throughout the culture period. Immunoprécipitation Assay Molt3/HIV-l,||B cells were labeled with [35S]methionine and immunoprecipitated by the monoclonal an tibody and by H IV-l-antibody-positive human serum as described elsewhere [16]. Monoclonal Antibody Preparation BALB/c mice (Charles River Breeding Labora tories), were immunized by successive intraperitoneal inoculations of 20 pg purified gpl20 from HTLV-1IIB emulsified in complete Freund’s adjuvant for the first inoculation and in incomplete Freund's adjuvant for the following five boosters (10 ug of protein each time), given 1 week apart. Three days after a final intraperito neal booster with 20 gg gpl20 in phosphate-buffered saline, splenic lymphocytes were fused with the NS-1 mouse myeloma cell line. The fusion procedure, cell culturing, determination of immunoglobulin subclass, and cloning of hybridoma lines were very similar to procedures previously described in the literature [17]. Mouse ascitic fluid containing monoclonal antibodies was prepared as described previously [17],
87
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/5/2018 9:49:50 PM
cessible to the immune system. Antibodies ca pable of neutralizing HIV-I infection of target cells are usually found in serum from HI V-l-infected individuals, albeit in low concentrations relative to those induced by other viruses [3-5]. Most of these antibodies bind gpl20 and neu tralize more than one serologically distinct iso late of the virus [5], In addition, native gpl20 and recombinant gpl60 and gpl20 have been shown to elicit type-specific antibodies that neutralize the infectivity of HIV-1 in vitro [5,6]. The major neutralizing epitope has been nar rowed down to a 24-amino-acid segment from amino acids 302 to 325 (V3 loop) [5,6]. Murine monoclonal antibodies directed towards this region blocked syncytia formation and neu tralized infectivity of HIV-1 [reviewed in ref. 5,6]. The major pathway of infection of T cells by HIV-1 involves pH-independent fusion of viral and host cell membranes [7, 8], This process is initiated by the interaction of gpl20 with its receptor, CD4 [9-11]. After gpl20 has bound to CD4, a conformational change is induced on the glycoprotein molecule which enhances the susceptibility of the V3 loop to protease diges tion, and eventually dissociates gpl20 from the viral membrane [12]. This results in the expo sure of the amino terminal hydrophobic do main of gp41 which initiates the fusion process [13,14]. In this communication, an HIV-1-antibodypositive human serum and a monoclonal anti body directed towards the external envelope glycoprotein have been used to further define the neutralizing epitope in the gpl20 molecule. Moreover, the monoclonal antibody was shown to inhibit a post-binding event during HIV-1 infection, indicated by its blocking the cleavage of the V3 loop by an exogenous pro tease.
Results
88
Fig. 1. Reactivity of M77 and PS12 with HIV-l pro teins. Molt3/HIV-lmB cells were labeled with [,5S]methionine for 7 h and the clarified lysate was immunoprecipitated with M77 (A, lane I) and PS12 (B. lane I) as described in Materials and Methods. For tunicamycin treatment the cells were pretreated for I h, labeled for 7 h in the presence of 1.5 pg/ml of tunicamycin and immunoprecipitated with M77 (A, lane 2) and PSI2 (B, lane 2).
of gpl20, as it was found to react with the deglycosylated 80-kD protein generated in the infected cell by tunicamycin treatment (fig. IB, lane 2). Biological Activity o f Monoclonal Antibody M77 and Human Serum PS12 Purified IgG from M77 was tested for syncy tia-blocking activity and for virus neutraliza tion. For the syncytia-blocking assay, the OEM cells were cocultured with Molt3/HIV-ImB cells in the presence of the antibody. Nearly 90% of the inhibition of syncytia was achieved at a concentration
