INFzCTION AND IMMUNITY, Apr. 1977, p. 9-11 Copyright © 1977 American Society for Microbiology
Vol. 16, No. 1 Printed in U.S.A.
Protection Against Herpes Simplex Virus Infection in Mice by
Corynebacterium parvum HOLGER KIRCHNER,* HANS MARTIN HIRT, AND KLAUS MUNK Institute of Virology, German Cancer Research Center, 6900 Heidelberg, Federal Republic of Germany Received for publication 25 October 1976
Corynebacterium parvum administered in mice prior to herpes simplex virus (HSV) infection significantly protected them against lethal encephalitis. This was seen both with a mouse strain highly susceptible to HSV and with one relatively resistant to HSV. Mice immunosuppressed by cyclophosphamide and showing an increased mortality after HSV infection were also protected by C. parvum
pretreatment. However, C. parvum given simultaneously with
or
after
HSV infection did not exert a therapeutic effect. Infections with herpes simplex virus (HSV) are ubiquitous in humans and usually are locally restricted. However, they may generalize in immuno-incompetent newborns and in immunodeficient or immunosuppressed patients (for review, see references 8 and 12). HSV is a common cause of fatal, sporadic encephalitis (2). HSV is also suspected to be an oncogenic agent, particularly in the development of cervical carcinoma (9). A satisfactory therapy for HSV infection does not exist, and attempts to specifically immunize against HSV have not been encouraging in terms of effectiveness (4). Therefore, it appears useful to screen nonspecific stimulants of the immune system for their antiviral activity. A mouse modeI of HSV-induced encephalitis has been previously used to study the effect of immunostimulants on HSV infection (11). In the present investigation we have tested killed Corynebacterium parvum, an adjuvant that has received great interest because of its antitumoral effects in animal systems but has been little studied for antiviral protection (10).
the supernatant was 3.5 x 106 plaque-forming units (PFU)/ml when assayed in cultures of primary mouse embryo fibroblasts. The 50% lethal dose of HSV-1 in STU mice after intraperitoneal (i.p.) infection, as determined by the Spearman-Karber method, was 2 x 101 PFU, whereas it was 5 x 105 PFU in B6 mice. Formalin-killed C. parvum (CN 6134, Ba 3935/A; kindly provided by M. T. Scott, Department of Experimental Immunobiology, The
Wellcome Research Laboratories, Beckenham, Kent, England) was injected i.p. at various times prior to and after HSV-1 infection.
RESULTS In initial experiments, we have found that maximal protection occurred when C. parvum was given 6 to 8 days before HSV-1 infection and protection could be demonstrated with HSV-1 doses up to 100 50% lethal doses in STU mice. No protection was seen when C. parvum was given on the day of viral infection or when it was tested therapeutically after infection. In experiment A of Table 1 the effect of 210 ,ug of C. parvum given 7 days prior to the injection of 103 PFU of HSV-1 in STU mice is shown. Whereas 19 of 20 mice injected with virus alone died after 5 to 10 days with clinical signs of encephalitis, only 2 of 20 mice died in the group pretreated with C. parvum. Similar results were obtained in experiment B of Table 1. In both experiments the differences between experimental and control groups were significant at the P < 0.001 level. B6 mice, in accordance with previous data of others (6), were found to be considerably more resistant to i.p. infection with HSV-1 than were STU mice. When B6 mice were injected with 106 PFU of HSV-1, 9 of 19 mice died (experiment C, Table 1). In the experimental group pretreated with 210 ,ug of C. parvum 7 days earlier, only 1 of 25 mice died
MATERIALS AND METHODS Inbred STU mice, originally obtained in 1961 from W. Schiifer, Tuibingen, Federal Republic of Germany, were bred in our department by continuous brother-sister mating. C57BL/6J BOM (B6) mice were obtained from GI. Bomholtgard, Ltd., Denmark. The weight of all mice was between 20 and 25 g, and they were 8 to 12 weeks of age when used in the experiments. HSV-1 (strain WAL) (7) has been adapted to mice by intracerebral passage in STU mice, and brain passage 35 was used in this study. Infected brains were homogenized in saline to obtain a 10% solution and stored at - 70°C. This suspension was regarded as a 10-1 dilution in the titrations. Immediately before injection, the suspension was centrifuged to remove cellular material. The titer of 9
10
KIRCHNER, HIRT, AND MUNK TABLE 1. Effect of pretreatment with killed C. parvum on HSV-1 infection in mice No. of survivers/groupa Expt
A B C
Control group injected with HSV-1
2/20 3/20 10/19
Experimental group injected with C. parvum
19/20t 14/20b 24/25C
Deaths usually occurred 5 to 10 days postinfection, and experiments were terminated after 21 days. C. parvum (210 pg/mouse) was injected i.p. 7 days before i.p. infection with HSV-1 (WAL). STU mice (experiments A and B) and C57BL/6 mice (experiment C) were infected with 103 and 106 PFU of HSV-1, respectively. b Significant differences between control group and experimental group by the chi-square test (P < 0.001). C p < 0.005. a
(difference significant at the P < 0.005 level). Mice surviving viral infection after pretreatment with C. parvum were found to be immune to a second viral challenge (data not shown). Treatment of B6 mice with 6 mg of cyclophosphamide (CY) on the day of HSV infection decreased the 50% lethal dose about 100-fold (Table 2). Mice pretreated with C. parvum 7 days previously were resistant to HSV given together with CY.
DISCUSSION Systemic administration of C. parvum has been shown to have a variety of biological effects, including an adjuvant effect on humoral immunity, a stimulation of antitumor cellular immunity, and induction of increased antibacterial resistance (for review, see references 3 and 10). There have been little data on the effects of C. parvum on viral infections. Halpern and co-workers (3) have reported protective effects of C. parvum on Mengo virus infection in mice. In our experiments, we have shown a striking protective effect of C. parvum on HSV-1-induced encephalitis in two strains of mice, one that is highly susceptible to i.p. infection and one that is relatively resistant. In view of the severe complications of HSV infections seen in immunosuppressed patients (8), we believe that our demonstration of a protective effect of C. parvum on HSV infection of CYtreated mice is particularly noteworthy. The mechanisms by which C. parvum exerts its antiviral effect are still to be identified. However, in view of the large body of evidence showing that much of the antitumor effect of C. parvum is mediated through general activation
INFECT. IMMUN.
TABLE 2. Effect of pretreatment with C. parvum on HSV-1 infections of B6 mice immunosuppressed by CY No. of survivors/group
Treatment Expt A
Expt B
HSV-1 alone 10/10 9/10 CY alone 10/10 10/10 HSV-1 plus CY 0/10 1/10 HSV-1 plus CY after pre9/10 9/10 treatment with C. parvum a a Mice were pretreated on day -7 with 210 ,g of C. parvum and on day 0 with 6 mg of CY and 103 PFU of HSV-1 (WAL).
of macrophages (10), it is reasonable to speculate that the antiviral effect is mediated by a similar mechanism. In many animal models a role of the macrophage in antiviral defense has been documented (1), particularly in studies of HSV-induced lethal encephalitis of mice (13). Preliminary evidence from our laboratory has indicated that spleen cells from C. parvuminjected mice produce increased levels of interferon (unpublished data), a mechanism that, in conjunction with macrophage activation, might be also operative in C. parvum-induced protection. Data similar to ours have appeared during the completion of our experiments. In these, the effect of several "immunostimulants" on HSV2-induced infection of newborn mice was studied (11). Among these substances (C. parvum was not tested), only BCG had a protective effect. Similarly, as in our study, only protective and not therapeutic effects were seen. Thus, it might be premature to suggest the use of C. parvum, which has been widely used in immunotherapy of human tumors (5), for antiviral clinical trials in humans. However, it has to be realized that in the animal model used here, the time required for a strong systemic activation by C. parvum is almost equivalent to the interval at which the animals start to die. In a clinical situation where infection is less fulminant, a positive effect still might be observed. ACKNOWLEDGMENTS We are most grateful to K. Keyssner and C. Kleinicke for their expert technical assistance and to D. Baumgartl for his cooperation in the animal experiments. LITERATURE CITED 1. Allison, A. C. 1974. On the role of mononuclear phagocytes in immunity against viruses. Prog. Med. Virol. 18:15-31. 2. Editorial. 1967. Encephalitis caused by herpes-simplex virus. N. Engl. J. Med. 277:1315-1316.
HSV INFECTION AND C. PARVUM
VOL. 16, 1977 3. Halpern, B., A. Fray, Y. Crepin, 0. Platica, A. M. Lorinet, A. Rabourdin, L. Sparros, and R. Isac. 1973. Corynebacterium parvum, a potent immunostimulant in experimental infections and in malignancies, p. 217-236. In G. E. W. Wolstenholm and J. Knight (ed.), Immunopotentiation, Ciba Foundation Symp. 18 (new series). Associated Scientific Publishers, London. 4. Hilleman, M. R. 1976. Herpes simplex vaccines. Cancer Res. 36:857-858. 5. Israel, L., and B. N. Halpern. 1972. Le Corynebacterium parvum dans les cancers avanc6s. Nouv. Presse Med. 1:19-23. 6. Lopez, C. 1975. Genetics of natural resistance to herpes-virus in mice. Nature (London) 258:152-153. 7. Munk, K., and D. Donner. 1963. Cytopathischer Effekt und Plaque Morphologie verschiedener Herpes-simplex Virus-stimme. Arch. Gosamte Virusforsch. 13:529-540. 8. Nahmias, A. J., S. L. Shore, S. Kohl, S. E. Starr, and
9. 10. 11.
12. 13.
11
R. B. Ashman. 1976. Immunology of herpes simplex virus infection: relevance to herpes simplex virus vaccines and cervical cancer. Cancer Res. 36:836-844. Rapp, F. 1973. Question: Do herpesviruses cause cancer? Answer: Of course they do! J. Natl. Cancer Inst. 50:825-832. Scott, M. T. 1974. Corynebacterium parvum as an immunotherapeutic anticancer agent. Semin. Oncol. 1:367-378. Starr, S. E., A. 1. Visintine, M. O. Tomeh, and A. J. Nahmias. 1976. Effects of immunostimulants on resistance of newborn mice to herpes simplex type 2 infection. Proc. Soc. Exp. Biol. Med. 152:57-60. Wheeler, C. E. 1975. Pathogenesis of recurrent herpes simplex infections. J. Invest. Dermatol. 65:341-346. Zisman, B., M. S. Hirsch, and A. C. Allison. 1969. Selective effects of anti-macrophage serum, silica, and anti-lymphocyte serum on pathogenesis of herpes virus infection on young adult mice. J. Immunol. 104:1155-1159.
Vol. 16, No. 1 Printed in U.S.A.
Protection Against Herpes Simplex Virus Infection in Mice by
Corynebacterium parvum HOLGER KIRCHNER,* HANS MARTIN HIRT, AND KLAUS MUNK Institute of Virology, German Cancer Research Center, 6900 Heidelberg, Federal Republic of Germany Received for publication 25 October 1976
Corynebacterium parvum administered in mice prior to herpes simplex virus (HSV) infection significantly protected them against lethal encephalitis. This was seen both with a mouse strain highly susceptible to HSV and with one relatively resistant to HSV. Mice immunosuppressed by cyclophosphamide and showing an increased mortality after HSV infection were also protected by C. parvum
pretreatment. However, C. parvum given simultaneously with
or
after
HSV infection did not exert a therapeutic effect. Infections with herpes simplex virus (HSV) are ubiquitous in humans and usually are locally restricted. However, they may generalize in immuno-incompetent newborns and in immunodeficient or immunosuppressed patients (for review, see references 8 and 12). HSV is a common cause of fatal, sporadic encephalitis (2). HSV is also suspected to be an oncogenic agent, particularly in the development of cervical carcinoma (9). A satisfactory therapy for HSV infection does not exist, and attempts to specifically immunize against HSV have not been encouraging in terms of effectiveness (4). Therefore, it appears useful to screen nonspecific stimulants of the immune system for their antiviral activity. A mouse modeI of HSV-induced encephalitis has been previously used to study the effect of immunostimulants on HSV infection (11). In the present investigation we have tested killed Corynebacterium parvum, an adjuvant that has received great interest because of its antitumoral effects in animal systems but has been little studied for antiviral protection (10).
the supernatant was 3.5 x 106 plaque-forming units (PFU)/ml when assayed in cultures of primary mouse embryo fibroblasts. The 50% lethal dose of HSV-1 in STU mice after intraperitoneal (i.p.) infection, as determined by the Spearman-Karber method, was 2 x 101 PFU, whereas it was 5 x 105 PFU in B6 mice. Formalin-killed C. parvum (CN 6134, Ba 3935/A; kindly provided by M. T. Scott, Department of Experimental Immunobiology, The
Wellcome Research Laboratories, Beckenham, Kent, England) was injected i.p. at various times prior to and after HSV-1 infection.
RESULTS In initial experiments, we have found that maximal protection occurred when C. parvum was given 6 to 8 days before HSV-1 infection and protection could be demonstrated with HSV-1 doses up to 100 50% lethal doses in STU mice. No protection was seen when C. parvum was given on the day of viral infection or when it was tested therapeutically after infection. In experiment A of Table 1 the effect of 210 ,ug of C. parvum given 7 days prior to the injection of 103 PFU of HSV-1 in STU mice is shown. Whereas 19 of 20 mice injected with virus alone died after 5 to 10 days with clinical signs of encephalitis, only 2 of 20 mice died in the group pretreated with C. parvum. Similar results were obtained in experiment B of Table 1. In both experiments the differences between experimental and control groups were significant at the P < 0.001 level. B6 mice, in accordance with previous data of others (6), were found to be considerably more resistant to i.p. infection with HSV-1 than were STU mice. When B6 mice were injected with 106 PFU of HSV-1, 9 of 19 mice died (experiment C, Table 1). In the experimental group pretreated with 210 ,ug of C. parvum 7 days earlier, only 1 of 25 mice died
MATERIALS AND METHODS Inbred STU mice, originally obtained in 1961 from W. Schiifer, Tuibingen, Federal Republic of Germany, were bred in our department by continuous brother-sister mating. C57BL/6J BOM (B6) mice were obtained from GI. Bomholtgard, Ltd., Denmark. The weight of all mice was between 20 and 25 g, and they were 8 to 12 weeks of age when used in the experiments. HSV-1 (strain WAL) (7) has been adapted to mice by intracerebral passage in STU mice, and brain passage 35 was used in this study. Infected brains were homogenized in saline to obtain a 10% solution and stored at - 70°C. This suspension was regarded as a 10-1 dilution in the titrations. Immediately before injection, the suspension was centrifuged to remove cellular material. The titer of 9
10
KIRCHNER, HIRT, AND MUNK TABLE 1. Effect of pretreatment with killed C. parvum on HSV-1 infection in mice No. of survivers/groupa Expt
A B C
Control group injected with HSV-1
2/20 3/20 10/19
Experimental group injected with C. parvum
19/20t 14/20b 24/25C
Deaths usually occurred 5 to 10 days postinfection, and experiments were terminated after 21 days. C. parvum (210 pg/mouse) was injected i.p. 7 days before i.p. infection with HSV-1 (WAL). STU mice (experiments A and B) and C57BL/6 mice (experiment C) were infected with 103 and 106 PFU of HSV-1, respectively. b Significant differences between control group and experimental group by the chi-square test (P < 0.001). C p < 0.005. a
(difference significant at the P < 0.005 level). Mice surviving viral infection after pretreatment with C. parvum were found to be immune to a second viral challenge (data not shown). Treatment of B6 mice with 6 mg of cyclophosphamide (CY) on the day of HSV infection decreased the 50% lethal dose about 100-fold (Table 2). Mice pretreated with C. parvum 7 days previously were resistant to HSV given together with CY.
DISCUSSION Systemic administration of C. parvum has been shown to have a variety of biological effects, including an adjuvant effect on humoral immunity, a stimulation of antitumor cellular immunity, and induction of increased antibacterial resistance (for review, see references 3 and 10). There have been little data on the effects of C. parvum on viral infections. Halpern and co-workers (3) have reported protective effects of C. parvum on Mengo virus infection in mice. In our experiments, we have shown a striking protective effect of C. parvum on HSV-1-induced encephalitis in two strains of mice, one that is highly susceptible to i.p. infection and one that is relatively resistant. In view of the severe complications of HSV infections seen in immunosuppressed patients (8), we believe that our demonstration of a protective effect of C. parvum on HSV infection of CYtreated mice is particularly noteworthy. The mechanisms by which C. parvum exerts its antiviral effect are still to be identified. However, in view of the large body of evidence showing that much of the antitumor effect of C. parvum is mediated through general activation
INFECT. IMMUN.
TABLE 2. Effect of pretreatment with C. parvum on HSV-1 infections of B6 mice immunosuppressed by CY No. of survivors/group
Treatment Expt A
Expt B
HSV-1 alone 10/10 9/10 CY alone 10/10 10/10 HSV-1 plus CY 0/10 1/10 HSV-1 plus CY after pre9/10 9/10 treatment with C. parvum a a Mice were pretreated on day -7 with 210 ,g of C. parvum and on day 0 with 6 mg of CY and 103 PFU of HSV-1 (WAL).
of macrophages (10), it is reasonable to speculate that the antiviral effect is mediated by a similar mechanism. In many animal models a role of the macrophage in antiviral defense has been documented (1), particularly in studies of HSV-induced lethal encephalitis of mice (13). Preliminary evidence from our laboratory has indicated that spleen cells from C. parvuminjected mice produce increased levels of interferon (unpublished data), a mechanism that, in conjunction with macrophage activation, might be also operative in C. parvum-induced protection. Data similar to ours have appeared during the completion of our experiments. In these, the effect of several "immunostimulants" on HSV2-induced infection of newborn mice was studied (11). Among these substances (C. parvum was not tested), only BCG had a protective effect. Similarly, as in our study, only protective and not therapeutic effects were seen. Thus, it might be premature to suggest the use of C. parvum, which has been widely used in immunotherapy of human tumors (5), for antiviral clinical trials in humans. However, it has to be realized that in the animal model used here, the time required for a strong systemic activation by C. parvum is almost equivalent to the interval at which the animals start to die. In a clinical situation where infection is less fulminant, a positive effect still might be observed. ACKNOWLEDGMENTS We are most grateful to K. Keyssner and C. Kleinicke for their expert technical assistance and to D. Baumgartl for his cooperation in the animal experiments. LITERATURE CITED 1. Allison, A. C. 1974. On the role of mononuclear phagocytes in immunity against viruses. Prog. Med. Virol. 18:15-31. 2. Editorial. 1967. Encephalitis caused by herpes-simplex virus. N. Engl. J. Med. 277:1315-1316.
HSV INFECTION AND C. PARVUM
VOL. 16, 1977 3. Halpern, B., A. Fray, Y. Crepin, 0. Platica, A. M. Lorinet, A. Rabourdin, L. Sparros, and R. Isac. 1973. Corynebacterium parvum, a potent immunostimulant in experimental infections and in malignancies, p. 217-236. In G. E. W. Wolstenholm and J. Knight (ed.), Immunopotentiation, Ciba Foundation Symp. 18 (new series). Associated Scientific Publishers, London. 4. Hilleman, M. R. 1976. Herpes simplex vaccines. Cancer Res. 36:857-858. 5. Israel, L., and B. N. Halpern. 1972. Le Corynebacterium parvum dans les cancers avanc6s. Nouv. Presse Med. 1:19-23. 6. Lopez, C. 1975. Genetics of natural resistance to herpes-virus in mice. Nature (London) 258:152-153. 7. Munk, K., and D. Donner. 1963. Cytopathischer Effekt und Plaque Morphologie verschiedener Herpes-simplex Virus-stimme. Arch. Gosamte Virusforsch. 13:529-540. 8. Nahmias, A. J., S. L. Shore, S. Kohl, S. E. Starr, and
9. 10. 11.
12. 13.
11
R. B. Ashman. 1976. Immunology of herpes simplex virus infection: relevance to herpes simplex virus vaccines and cervical cancer. Cancer Res. 36:836-844. Rapp, F. 1973. Question: Do herpesviruses cause cancer? Answer: Of course they do! J. Natl. Cancer Inst. 50:825-832. Scott, M. T. 1974. Corynebacterium parvum as an immunotherapeutic anticancer agent. Semin. Oncol. 1:367-378. Starr, S. E., A. 1. Visintine, M. O. Tomeh, and A. J. Nahmias. 1976. Effects of immunostimulants on resistance of newborn mice to herpes simplex type 2 infection. Proc. Soc. Exp. Biol. Med. 152:57-60. Wheeler, C. E. 1975. Pathogenesis of recurrent herpes simplex infections. J. Invest. Dermatol. 65:341-346. Zisman, B., M. S. Hirsch, and A. C. Allison. 1969. Selective effects of anti-macrophage serum, silica, and anti-lymphocyte serum on pathogenesis of herpes virus infection on young adult mice. J. Immunol. 104:1155-1159.
