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Intervirology 1991;32:351-360
Different Mechanisms of Protection by Monoclonal and Polyclonal Antibodies during the Course of Herpes Simplex Virus Infection Anna M. Eis-Hiibinger, Klaudia Mohr, Karl E. Schneweis Institute of Medical Microbiology and Immunology, University of Bonn, FRG
Keywords. Herpes simplex virus • Monoclonal antibodies • Polyclonal immune serum ■ Protection mechanisms • Mouse model
Introduction It is well established that virus-specific immune serum induces premature latency of herpes simplex virus (HSV) in the dorsal root ganglia of infected mice [1, 2], Furthermore, Address inquiries to: Dr. Anna M. Eis-Hiibinger, Institut für Medizinische Mikrobiologie und Immu nologie, Universität Bonn. Sigmund-Freud-Strasse 25, D-W-5300 Bonn I (FRG) Received: July 30, 1990 Accepted: November 14,1990
the number of infected ganglia is consider ably decreased if immune serum is given soon after virus inoculation, although virus multiplication at the inoculation site is not depressed [2-4], Therefore, it has been assumed that anti bodies are effective at the stage of ganglionic infection. By this mechanism, the spread of virus from the peripheral site of infection to the central nervous system is interrupted, and the mice are protected against lethal enceph alitis. For that reason, passive administration of immune serum is discussed for treatment
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:43:53 AM
Summary. Young adult C57BL mice intravaginally inoculated with herpes simplex virus type I and passively immunized with a monoclonal antibody directed against herpes simplex virus type I glycoprotein gB were shown to be more effectively protected against infection as compared with mice treated with polyclonal immune serum. In contrast to polyclonal antiserum, the monoclonal antibody markedly restricted viral multiplication in the infected mucous membranes. Consequently, skin lesions were completely prevented, and the extent of ganglionic infection was significantly reduced. The mechanism by which a monoclonal antibody, specific to glycoprotein gC, effected protection also differed from that of the hyperimmune serum, since premature latency was not induced. The data provide strong evidence that the mechanisms of protection mediated by antibodies depend on their epitope specificity. The inhibition of active antibody response after passive immunization was inducible by polyclonal antibody only, not by monoclonal antibodies.
352
Eis-Hubinger/Mohr/Schneweis
1
2
3
4
5
6
Materials and Methods Virus HSV type 1(HSV-1) strains WAL wt and 342 hv [7] were grown and titrated in Vero cells. Growth me dium consisted of Eagle's MEM supplemented with 10% fetal calf serum. Virus was released from infected cells by sonication, and the lysate was clarified by centrifugation at 1,500 g for 30 min. Viral titers were determined by evaluating the I Dso/0.05 ml in microti ter plates on Vero cells according to the method of Reed and Muench [8].
of severe HSV infections, especially in com bination with a specific antiviral chemother apeutic agent [5, 6]. In this report we describe two monoclonal antibodies (Mabs), specific for glycoprotein gB and gC, respectively, exhibiting different mechanisms of protection from that induced by polyclonal immune serum.
Mice Female C57BL/6J mice, 5-7 weeks old, were ob tained from the Zentralinstitut fiir Versuchstiere, Hannover, FRG. The mice weighed between 15 and 18 g at the time of infection. Antibodies Mabs were prepared as described by Holland et al. [9], i.e., by fusing spleen cells from BALB/c mice, hyperimmunized with HSV-I strain 342 hv, with the myeloma cell line X63-Ag8.653 (obtained from Lothar Stitz, University of Giessen, FRG) by adding polyethylene glycol. Hybrids were selected in HAT medium. Cell-free culture fluid obtained from hy bridoma cultures, grown in serum-free Iscove’s me dium, served as the source of Mabs, then concen trated by ammonium sulfate precipitation followed by dialysis. For control, equivalent volumes of Is cove’s medium were treated in the same manner. The specificities of the Mabs 2c and III E8 were deter mined by immunoblotting using a biotin-streptav idin-peroxidase detection system [10-13] and viral ly sate prepared from sucrose gradient purified HSV-1 [14]. Mab 2c was determined to be specific for HSV-1 glycoprotein B, Mab III E8 for HSV-1 glycoprotein C (fig. 1). Mab 1was not specific for HSV - therefore, an irrelevant antibody. The isotypes of the Mabs were determined by standard double-diffusion agarose gels using rabbit antimouse immunoglobulin class and subclass sera. Mab 2c and Mab 111 E8 belonged to the immunoglob ulin G (IgG) 2a subclass, Mab 1 to the IgG class, subclass not known. The human standard immune serum preparation used (Beriglobin S; Behring, FRG), containing 160 mg of IgG per milliliter, had a complement-inde pendent neutralizing titer of 1:1,280 to HSV-1 in
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:43:53 AM
Fig. I. Immunoblot with the Mabs 2c and III E8. Vero cells were infected either with HSV-I, mutant strain GCI-8, which is deleted for glycoprotein gC and glycoprotein gE [27] (lanes 2 and 5), HSV-1, mut ant strain A NG E3/3, deleted for glycoprotein gE [28] (lanes 3 and 6), or HSV-1, strain 342 hv [7] (lane 4). Virus was collected from culture supernatants and infected cells, clarified by sucrose gradient, lysed, and subjected to SDS-PAGE. After electroblotting to ni trocellulose, strips were incubated with hybridoma culture fluid, and the complexes were detected with a biotin-streptavidin-peroxidase detection system. Lane 1: molecular weight markers ( x 10J): lanes 2-4: Mab 2c, recognizing the mature form of glycoprotein gB and the precursor pgB [29]; lanes 5 and 6: Mab 111 E8, recognizing glycoprotein gC, its precursor pgC, and regularly a third gC-l-specific band which was also found by Zweig et al. [30] and designated as 65,000-molecular-weight degradation product. This band was also consistently detected by Mab III E8 when we used cell extracts and culture supernatants of clones derived from NIH 3T3 cells transfected with the gC-1 gene [31]. The mutant strains were kindly provided by Prof. H.C. Kaerner, Heidelberg, FRG.
Mechanisms of Protection by Monoclonal Antibodies against HSV Infection
Neutralization Test The neutralization test was performed on Vero cells in microtiter plates. 0.025 ml of diluted antibody solutions or sera was incubated with 100 TC1DS0 of HSV-1, with and without guinea pig complement, diluted 1:10. Titers were calculated on the basis of the highest serum dilution preventing virus-induced CPE in 50% of the cultures and expressed as reciprocals of the dilutions [16]. Mouse Protection Experiments Animal procedures were performed as described elsewhere [2, 17]. Briefly, the mice were inoculated intravaginally with 2 x 1060 ID
Intervirology 1991;32:351-360
Different Mechanisms of Protection by Monoclonal and Polyclonal Antibodies during the Course of Herpes Simplex Virus Infection Anna M. Eis-Hiibinger, Klaudia Mohr, Karl E. Schneweis Institute of Medical Microbiology and Immunology, University of Bonn, FRG
Keywords. Herpes simplex virus • Monoclonal antibodies • Polyclonal immune serum ■ Protection mechanisms • Mouse model
Introduction It is well established that virus-specific immune serum induces premature latency of herpes simplex virus (HSV) in the dorsal root ganglia of infected mice [1, 2], Furthermore, Address inquiries to: Dr. Anna M. Eis-Hiibinger, Institut für Medizinische Mikrobiologie und Immu nologie, Universität Bonn. Sigmund-Freud-Strasse 25, D-W-5300 Bonn I (FRG) Received: July 30, 1990 Accepted: November 14,1990
the number of infected ganglia is consider ably decreased if immune serum is given soon after virus inoculation, although virus multiplication at the inoculation site is not depressed [2-4], Therefore, it has been assumed that anti bodies are effective at the stage of ganglionic infection. By this mechanism, the spread of virus from the peripheral site of infection to the central nervous system is interrupted, and the mice are protected against lethal enceph alitis. For that reason, passive administration of immune serum is discussed for treatment
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:43:53 AM
Summary. Young adult C57BL mice intravaginally inoculated with herpes simplex virus type I and passively immunized with a monoclonal antibody directed against herpes simplex virus type I glycoprotein gB were shown to be more effectively protected against infection as compared with mice treated with polyclonal immune serum. In contrast to polyclonal antiserum, the monoclonal antibody markedly restricted viral multiplication in the infected mucous membranes. Consequently, skin lesions were completely prevented, and the extent of ganglionic infection was significantly reduced. The mechanism by which a monoclonal antibody, specific to glycoprotein gC, effected protection also differed from that of the hyperimmune serum, since premature latency was not induced. The data provide strong evidence that the mechanisms of protection mediated by antibodies depend on their epitope specificity. The inhibition of active antibody response after passive immunization was inducible by polyclonal antibody only, not by monoclonal antibodies.
352
Eis-Hubinger/Mohr/Schneweis
1
2
3
4
5
6
Materials and Methods Virus HSV type 1(HSV-1) strains WAL wt and 342 hv [7] were grown and titrated in Vero cells. Growth me dium consisted of Eagle's MEM supplemented with 10% fetal calf serum. Virus was released from infected cells by sonication, and the lysate was clarified by centrifugation at 1,500 g for 30 min. Viral titers were determined by evaluating the I Dso/0.05 ml in microti ter plates on Vero cells according to the method of Reed and Muench [8].
of severe HSV infections, especially in com bination with a specific antiviral chemother apeutic agent [5, 6]. In this report we describe two monoclonal antibodies (Mabs), specific for glycoprotein gB and gC, respectively, exhibiting different mechanisms of protection from that induced by polyclonal immune serum.
Mice Female C57BL/6J mice, 5-7 weeks old, were ob tained from the Zentralinstitut fiir Versuchstiere, Hannover, FRG. The mice weighed between 15 and 18 g at the time of infection. Antibodies Mabs were prepared as described by Holland et al. [9], i.e., by fusing spleen cells from BALB/c mice, hyperimmunized with HSV-I strain 342 hv, with the myeloma cell line X63-Ag8.653 (obtained from Lothar Stitz, University of Giessen, FRG) by adding polyethylene glycol. Hybrids were selected in HAT medium. Cell-free culture fluid obtained from hy bridoma cultures, grown in serum-free Iscove’s me dium, served as the source of Mabs, then concen trated by ammonium sulfate precipitation followed by dialysis. For control, equivalent volumes of Is cove’s medium were treated in the same manner. The specificities of the Mabs 2c and III E8 were deter mined by immunoblotting using a biotin-streptav idin-peroxidase detection system [10-13] and viral ly sate prepared from sucrose gradient purified HSV-1 [14]. Mab 2c was determined to be specific for HSV-1 glycoprotein B, Mab III E8 for HSV-1 glycoprotein C (fig. 1). Mab 1was not specific for HSV - therefore, an irrelevant antibody. The isotypes of the Mabs were determined by standard double-diffusion agarose gels using rabbit antimouse immunoglobulin class and subclass sera. Mab 2c and Mab 111 E8 belonged to the immunoglob ulin G (IgG) 2a subclass, Mab 1 to the IgG class, subclass not known. The human standard immune serum preparation used (Beriglobin S; Behring, FRG), containing 160 mg of IgG per milliliter, had a complement-inde pendent neutralizing titer of 1:1,280 to HSV-1 in
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 10:43:53 AM
Fig. I. Immunoblot with the Mabs 2c and III E8. Vero cells were infected either with HSV-I, mutant strain GCI-8, which is deleted for glycoprotein gC and glycoprotein gE [27] (lanes 2 and 5), HSV-1, mut ant strain A NG E3/3, deleted for glycoprotein gE [28] (lanes 3 and 6), or HSV-1, strain 342 hv [7] (lane 4). Virus was collected from culture supernatants and infected cells, clarified by sucrose gradient, lysed, and subjected to SDS-PAGE. After electroblotting to ni trocellulose, strips were incubated with hybridoma culture fluid, and the complexes were detected with a biotin-streptavidin-peroxidase detection system. Lane 1: molecular weight markers ( x 10J): lanes 2-4: Mab 2c, recognizing the mature form of glycoprotein gB and the precursor pgB [29]; lanes 5 and 6: Mab 111 E8, recognizing glycoprotein gC, its precursor pgC, and regularly a third gC-l-specific band which was also found by Zweig et al. [30] and designated as 65,000-molecular-weight degradation product. This band was also consistently detected by Mab III E8 when we used cell extracts and culture supernatants of clones derived from NIH 3T3 cells transfected with the gC-1 gene [31]. The mutant strains were kindly provided by Prof. H.C. Kaerner, Heidelberg, FRG.
Mechanisms of Protection by Monoclonal Antibodies against HSV Infection
Neutralization Test The neutralization test was performed on Vero cells in microtiter plates. 0.025 ml of diluted antibody solutions or sera was incubated with 100 TC1DS0 of HSV-1, with and without guinea pig complement, diluted 1:10. Titers were calculated on the basis of the highest serum dilution preventing virus-induced CPE in 50% of the cultures and expressed as reciprocals of the dilutions [16]. Mouse Protection Experiments Animal procedures were performed as described elsewhere [2, 17]. Briefly, the mice were inoculated intravaginally with 2 x 1060 ID
