- Email: [email protected]
or Acyclovir
Zbl. Bakt. 285, 445-449 (1997) © Gustav Fischer Verlag, Jena
Protection and Therapy of Experimental Herpesvirus Infections in Mice with Immunomodulating Propionibacterium avidum KP-40 and/or Acyclovir M. KOBUS\ M. LUCZAK], E. SOBICZEWSKA2, S. SZMIGIELSKI 2 , J. JELJASZEWICZ3, and G. PULVERER 4 Department of Virology, University Medical School, Warsaw, Poland Military Institute of Hygiene and Epidemiology, Warsaw, Poland 3 National Institute of H ygiene, Warsaw, Poland 4 Institute of Medical Microbiology and Hygiene, University of Cologne, Cologne, Ger many 1
2
Received April 13, 1996 . Revision received April 29, 1996 . Accepted May 17, 1996
Summary Protection and therapy of NMRI mice with experimental herpes virus (HSV-l) encephal itis were investigated using heat-killed, lyophilized Propionibacterium avidum KP-40 (PA) and/or the herpes-specific antiviral substance acyclovir (ACL) as immunomodifier. Poly I: C as a potent macrophage activator was used as a reference compound for PA. Survival of ex perimental HSV-l infections during 18 days following the inoculation of viruses was used for the evaluation of the effects of immunotherapy. The applied model of HSV-l infection resulted in a mortality of about 87% ofNMRI mice at 7-16 days following the inoculation of the virus. Treatment with ACL or Poly I: C at the day of HSV-1 infection resulted in a lowering of the mortality rate to about 40 % (p < 0.05) . PA applied 4 days before HSV-l infection lowered the mortality rate to 27% , while treatment 2 days after infection was less effective and the mortality rate reached 44%, although still being significantly lower (p < 0.01) than in untreated controls. A combined treatment with ACL and PA on the day of HSV-l infection protected 93 % of animals against the development of clinically detect able herpesvirus encephalitis.
Prevention and therapy of herpes virus infections still remains an unresolved prob lem (2) . Introduction of acyclovir (sodium 9[(2-hydroxyethoxy)-methylJ-guanine), a specific inhibitor of replication of herpesviruses, has improved the situation consider ably. But soon it became obvious that acyclovir-resistant herpesvirus infections occur quite frequently (5, 15). Therefore, newly synthesized inhibitors of herpesvirus repli cation are still being examined in therapeutic trials (2, 16, 20). On the other side, herpes virus infection is an established and widely used experimental model, the course of these infections being strongly related to the immune status of the host (4, 12, 13). It has been repeatedly demonstrated that a modulation of the host's immune status can
446
M. Kobus et al.
influence the course of herpesvirus infections (7, 8, 14) and may result in a better sur vival of otherwise lethal infections (9, 10,21). Some 20 years ago, anaerobic coryneforms possessing macrophage-activating prop erties have raised great interest because of their antitumoral effects in certain experi mental models (1). Moreover, pretreatment of mice with anaerobic propionibacteria resulted in a marked nonspecific protection against different viral infections (9, 22), including herpesvirus-induced encephalitis. It is known that there exist marked differ ences in the immunomodulating and antiviral properties of single strains within the ge nus Propionibacterium, some of the strains being totally inactive (1, 11). P. granulo sum KP-45 whole cells and isolated cell walls protected mice against herpesvirus en cephalitis when administered a few days prior to the inoculation of viruses (21). Sev eral years ago we introduced another immunomodulating strain, namely Propionibac terium avidum KP-40 (PA) because of its better standardization possibilities. Both propionibacterial strains used have been shown to possess pronounced immunostim ulating potency with a low immunogenicity and no toxicity in mice (11, 17, 19). All experimental and clinical observation data collected for PA strongly suggest that PA may be a useful immunomodulating agent in clinical medicine (for review, see 3, 17). It has been the aim of the present investigations to study the prophylactic and ther apeutic potency of Propionibacterium avidum KP-40 (PA) in experimental herpesvirus encephalitis in mice, in comparison to earlier successful studies with the bacterial im munomodifier, P. granulosum KP-45 (30). Materials and Methods 1. Mice: NMRI mice used for the infection with herpes simplex viruses were obtained from the Rega Institute of Virology (Catholic University of Leuven, Belgium) by the cour tesy of Prof. E. de Clercq and grown at the Department of Virology in Warsaw, Poland. Mice were bred under standard conditions and used for experiments at the age of 6 weeks. 2. Herpes simplex virus (HSV-1): HSV-l (strain KOS) was propagated in primary rabbit kidney (PRK) cell cultures. Stock virus had about 10 6 TCIDso/mL (Tissue Culture Infectious Dose) and was stored at -70°C. For the infection of mice, the stock virus was diluted 1:2 in Parker medium; 0.02 mL of the virus suspension, corresponding to 104 TCID so per mouse, was diluted in 0.2 mL of Ringer's solution and injected intra peritoneally. This resulted in the development of herpes virus encephalitis (confirmed by a high titre of HSV-1 in brain tissues during earlier studies of this infection) with about 85% of control animals dying between the 7th and the 16th day after the virus inoculation. Mortality was noted daily, animals surviving 18 days after in fection without signs of illness were counted as survivors. 3. Propionibacterium avidum KP-40 (PA): PA (heat-killed, lyophilized bacteria) was ob tained from the Institute of Medical Microbiology and Hygiene, University of Cologne, Ger many. The bacteria were grown, harvested and prepared as described earlier (11, 17). Be fore use, all samples of PA were tested under standard conditions for their immunostimu la ting potency (17). For the treatment of mice, a stock suspension of PA in double-distilled water containing 5 mg/mL was prepared 4-6 h before use and stored at 4 0C. The stock suspension of PA was diluted 1:2 with Ringer's solution and 0.2 mL of the dilution containing 0.5 mg of PA was injected intra peritoneally once per mouse. 4. Acyclovir (ACL): Sodium 9([2-hydroxyethoxy]-methyl)-guanine in form of lyophilized Zovirax (Wellcome, Great Britain) for intravenous administration in 250 mg vials was used throughout the experiments. For the treatment of mice, a stock solution of ACL in 0.15 M phosphate buffer pH 7.4 containing 1 mg/mL was prepared and 0.2 of this solution was in-
Herpes Virus and Immunomodulation
447
jected intraperitoneally twice daily (at 8.00 a.m. and 8.00 p.m.) during 10 days after the in fection with HSV-l (corresponding to 15 mg/kg b.w.). 5. Poly I: C: Polyriboinosinic polyribocitidylic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). For the treatment of mice, 0.2 mL of 200 ~g/mL solution of Po ly I: C in Ringer's medium (corresponding to 2.0 mg/kg b. w.) was injected intraperitoneal ly once daily for four days after the infection with HSV-1. 6. Experiments: Six-week-old male NMRI mice were randomly divided into 7 experi mental groups of 30 animals each (Table 1). All groups were infected intraperitoneally with 0.2 mL (104 TClD so ) of strain KOS HSV-l solution. Four days before the inoculation of vi ruses, on the day of infection or 2 days after HSV-l injection, the respective groups of mice (Table 1) were treated with Poly I: C, PA, ACL or a combination of these agents. Mortality was noted daily for 18 days following infection. 7. Statistical analysis: For analysis of the significance of differences in mortality between groups of animals, the chi-square test with Yates correction and a test for proportions (sta tistical calculator, CSS Statistica Win 4.5.) were used. In all cases, p < 0.05 was considered as significant.
Results and Discussion Inoculation of 104 TCID so of HSV-l resulted in death of 26 out of 30 (86.7%) of control mice (Table 1). The mortality started 7 days after infection, then grew rapidly and reached 50% of animals on the 10th day. Treatment with PA significantly lowered the mortality rate p < 0.01) (Table 1). Administration of ACL (15 mg/kg b.w.) on the day of HSV-l infection and on 10 consecutive days lowered the mortality rate to 36.7% (p < 0.01), but still about 113 of HSV-1 infected mice died within 18 days. The survi val of HSV-1 infections could be considerably increased by a combined treatment with ACL and PA. Administration of ACL for 10 days after HSV-1 infection combined with a single injection of PA on the day of virus inoculation resulted in a survival rate of 28 out of 30 mice (Table 1). This combination of treatment significantly enhanced the sur vival rate when compared not only to controls but also to the Poly I: C-treated group. The model of experimental HSV-l encephalitis in mice allows a valid testing of pro phylactic and therapeutic modalities. The course of experimental HSV-l infections in
Table 1. Survival rates of NMRI mice and results of different treatment schedules Group of mice (N
= 30)
Controls (untreated) Poly I:C 2 mg/kg (with HSV) PA 25 mg/kg, 4 days before HSV PA 25 mg/kg with HSV PA 25 mg/kg, 2 days after HSV ACL 15 mg/kg with HSV ACL + PA with HSV
18-day survival after HSV-l infection Number of mice surviving
Survival rate
4 18 22 21 17 19 28
0.133 0.60* 0.733* 0.70* 0.566* 0.633* 0.933*@
* p < 0.01 - compared to controls (untreated). @ p< 0.01 - compared to Poly I: C-treated group. PA = Propionibacterium avidum KP-40; ACL =Acyclovir.
448
M. Kobus et a1.
mice depends on the strain of animals, their age, the strain of viruses and the way of inoculation (6). In general, C 3H and BALB/c inbred mouse strains and most of their hybrids are considered as being relatively resistant to herpesviruses, while NMRI and Swiss mice belong to sensitive strains (18). NMRI mice were often challenged for ex perimental investigations of HSV-1 infections and testing of therapeutic modalitis (6, 21), however, in most of the earlier studies the course of experimental infections was very fast with nearly all animals dying within a few days after virus inoculation. For example, in our earlier study on the protection and therapy of experimental HSV-1 in fections in mice with Propionibacterium granulosum KP-45 (21), the intranasal infec tion of 12-day-old NMRI mice resulted in a mortality of 50% of control animals dur ing 4-5 days and death of 95% of mice within 9 days after infection. This rapid de velopment of disease left no time for modulations of the immune responses when pro pionibacteria were administered 3 days after the inoculation of viruses and, therefore, the mortality was only slightly lowered in this group. The model applied in the present study used older mice (6 weeks instead of 12 days), a different way of inoculation (intraperitoneal instead of intranasal) and a lower infective dose (10 4 TCID so ). These modalities resulted in a slower development of the disease, with all animals surviving the first 6 days after infection. Mortality started 7 days after virus inoculation and rap idly grew up to 16 days after infection, when about 85% of control animals had died. The bacterial immunomodifier PA tested in the present study exhibited prophylactic and therapeutic potency. PA administered either 4 days before or 2 days after the inoc ulation of viruses lowered the mortality of HSV-1-infected mice from 87% to 27-44% and prolonged survival of the animals. In the control group, mortality started 7 days af ter infection, while in PA-treated groups the first dying animals appeared a few days lat er (8-10 days after inoculation of viruses). In the present study, the best protection against mortality from herpes virus infections was obtained by the treatment of HSV-1infected mice with a combination of antiviral ACL and immunomodulation with PA. The treatment started on the day of virus inoculation and lasted during the first days af ter the infection, being therefore considered as a therapeutic modality with some clini cal relevance. The applied therapy protocol lowered the mortality rate from 87% in the controls to 7%, being significantly lower (p < 0.01) when compared with mortality rates of animals treated with ACL, PA or Poly I:C alone. ACL is a herpes-specific antiviral substance which inhibits the multiplication of virus particles and therefore limits the spread of the infection (16). In local infections with HSV (e. g. skin infections, keratitis), ACL totally inhibits virus replication and allows a cure of the infection (15). However, in general infections of sensitive hosts with large infective doses of viruses, only a par tial protection is possible (2). In the present model, the use of ACL at a dose of 15 mg/kg b. w. for 10 days after infection resulted in lowering the mortality from 87% to 37%. References 1. Adlam, C. and M. T. Scott: Lymphoreticular stimulatory properties of Corynebacterium parvum and related bacteria. J. Med. Microbiol. 6 (1973) 262-274 2. Balfour, H. H., C. Benson, J. Braun, B. Cassen, A. Erice, A. Friedman-Kien, T. Klein, B. Polsky, et a1.: Management of acyclovir-resistant herpes simplex and varicella zoster virus infections. J. Acquired Immun. Def. Syndr. 7 (1994) 254-260 3. Beuth, J., H. L. Ko, K. M. Peters, B. Bornhofen, and C. Pulverer: Behaviour of lympho cyte subsets in reponse to immunotherapy with Propionibacterium avidum KP-40 in can cer patients. Zb1. Bakt. 273 (1990) 386-390
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4. Catalano, L. w: and S. Baron: Protection against herpes virus and encephalomyocarditis virus encephalitis with a double stranded RNA inducer of interferon. Proc. Soc. Exp. BioI. Med. 133 (1970) 684-689 5. Collins, P. and M. N. Ellis: Sensitivity monitoring of clinical isolates of herpes simplex virus to acyclovir. J. Med. Virol. 41 (Suppl. 1) (1993) 58-66 6. De Clerq, E. and M. Luczak: Intranasal challenge of mice with herpes simplex virus: experimental model for evaluating the efficacy of antiviral drugs. J. Infect. Dis. 133 (Suppl.) (1976) A226-A236 7. Greene, J.J.: Double-stranded RNA and its analogs: The prospects and the promise of the first nucleic acid therapeutic agent. In: Clinical Applications of Interferon and IFN Inducers (ed.), Strangfellow, DA pp. 245-276, Marcel Dekker, Inc., New York (1986) 8. Ikeda, S., S. Nishiya, A. Yamamoto, T. Yamase, C. Nishimura, and E. De Clerq: Activity of the Keggin polyoxotungstate MP-19 against herpes simplex virus type 2 infection in immunosuppressed mice: role of peritoneal macrophage activation. J. Med. Virol. 41 (1993) 191-195 9. Kirchner, H., H. M. Hirt, and K. Munk: Protection against herpes simplex virus infec tion in mice by Corynebacterium parvum. Infect. Immun. 16 (1977) 9-11 10. Kirchner, H., M. T. Scott, and K. Munk: Protection of mice against viral infections by Corynebacterium parvum and Bordetella pertussis. J. Gen. Virol. 41 (1978) 97-104 11. Ko, H. L., w: Roszkowski, J. Jeljaszewicz, and G. Pulverer: Comparative study on the immunostimulatory potency of different propionibacteria strains. Med. Microbiol. Im munol. 170 (1981) 1-9 12. Mogensen, S. c.: Role of macrophage in natural resistance to virus infections. Micro bioI. Rev. 43 (1979) 1-34 13 . Morahan, P. S.: Interactions of herpes viruses with mononuclear phagocytes. In: Immu nobiology of Herpes Simplex Virus Infections, Rouse, B. T. and C. Lopez (eds.), pp. 7192, CRC Press, Boca Raton, FL (1984) 14. Morahan, P. S., E. R. Kern, and L. A. Glasgow: Immunomodulator-induced resistance against herpes simplex virus. Proc. Soc. Exp. BioI. Med. 154 (1977) 615-619 15. Nyquist, A. c., H. A. Rotbart, M. Cotton, C. Robinson, and A. Weinberg: Acyclovir resistant neonatal herpes simplex virus infection of the larynx. J. Pediatrics 124 (1994) 967-971 16. Pelosi, E., K. A. Hicks, S. L. Sacks, and D. M. Coen: Heterogenicity of a herpes simplex virus clinical isolate exhibiting resistance to acyclovir and foscarnet. Adv. Exp. Med. BioI. 312 (1992) 151-158 17. Pulverer, G., H. L. Ko, w: Roszkowski, K. Roszkowski, and J. Jeljaszewicz: Immuno modulation by propionibacteria. Clin. Immuno!. Newsletters 6 (1985) 51-54 18. Pyo, S., J. D. Gangemi, A. Ghaffar, and E. P. Mayer: Poly I: C-induced anti-herpes sim plex virus type 1 activity in inflammatory macro phages is mediated by induction of interferon-beta. J. Leukocyte BioI. 50 (1991) 479-487 19. Roszkowski, K. Roszkowski, H. L. Ko, J. Beuth, and J. Jeljaszewicz: Immunomodu Iation by propionibacteria. Zbl. Bakt. 274 (1990) 289-298 20. Safrin, S., T. Elbeik, and J. Mills: A rapid screen test for in vitro susceptibility of clinical herpes simplex virus isolates. J. Infect. Dis. 169 (1994) 879-882 21. Szmigielski, S., M. Kobus, J. Gil, J.Jeljaszewicz, and G. Pulverer: Protection and thera py of mice with acute and chronic experimental virus infections with Propionibacteri um granulosum KP-45. Zbl. Bakt. 248 (1980) 286-295 22. Szmigie/ski, S., M. Kobus, J. Gil, J.Jeljaszewicz, and G. Pulverer: Antineoplastic and antiviral properties of Propionibacterium granulosum. Drugs Exptl. Clin. Res. 8 (1982) 387-401
w.,
Prof. Dr. Dr. h. c. G. Pulverer, Inst. f. Med. Mikrobiologie und Hygiene der Universitat, Goldenfelsstrage 19-21, D-50935 Kaln, Germany
Protection and Therapy of Experimental Herpesvirus Infections in Mice with Immunomodulating Propionibacterium avidum KP-40 and/or Acyclovir M. KOBUS\ M. LUCZAK], E. SOBICZEWSKA2, S. SZMIGIELSKI 2 , J. JELJASZEWICZ3, and G. PULVERER 4 Department of Virology, University Medical School, Warsaw, Poland Military Institute of Hygiene and Epidemiology, Warsaw, Poland 3 National Institute of H ygiene, Warsaw, Poland 4 Institute of Medical Microbiology and Hygiene, University of Cologne, Cologne, Ger many 1
2
Received April 13, 1996 . Revision received April 29, 1996 . Accepted May 17, 1996
Summary Protection and therapy of NMRI mice with experimental herpes virus (HSV-l) encephal itis were investigated using heat-killed, lyophilized Propionibacterium avidum KP-40 (PA) and/or the herpes-specific antiviral substance acyclovir (ACL) as immunomodifier. Poly I: C as a potent macrophage activator was used as a reference compound for PA. Survival of ex perimental HSV-l infections during 18 days following the inoculation of viruses was used for the evaluation of the effects of immunotherapy. The applied model of HSV-l infection resulted in a mortality of about 87% ofNMRI mice at 7-16 days following the inoculation of the virus. Treatment with ACL or Poly I: C at the day of HSV-1 infection resulted in a lowering of the mortality rate to about 40 % (p < 0.05) . PA applied 4 days before HSV-l infection lowered the mortality rate to 27% , while treatment 2 days after infection was less effective and the mortality rate reached 44%, although still being significantly lower (p < 0.01) than in untreated controls. A combined treatment with ACL and PA on the day of HSV-l infection protected 93 % of animals against the development of clinically detect able herpesvirus encephalitis.
Prevention and therapy of herpes virus infections still remains an unresolved prob lem (2) . Introduction of acyclovir (sodium 9[(2-hydroxyethoxy)-methylJ-guanine), a specific inhibitor of replication of herpesviruses, has improved the situation consider ably. But soon it became obvious that acyclovir-resistant herpesvirus infections occur quite frequently (5, 15). Therefore, newly synthesized inhibitors of herpesvirus repli cation are still being examined in therapeutic trials (2, 16, 20). On the other side, herpes virus infection is an established and widely used experimental model, the course of these infections being strongly related to the immune status of the host (4, 12, 13). It has been repeatedly demonstrated that a modulation of the host's immune status can
446
M. Kobus et al.
influence the course of herpesvirus infections (7, 8, 14) and may result in a better sur vival of otherwise lethal infections (9, 10,21). Some 20 years ago, anaerobic coryneforms possessing macrophage-activating prop erties have raised great interest because of their antitumoral effects in certain experi mental models (1). Moreover, pretreatment of mice with anaerobic propionibacteria resulted in a marked nonspecific protection against different viral infections (9, 22), including herpesvirus-induced encephalitis. It is known that there exist marked differ ences in the immunomodulating and antiviral properties of single strains within the ge nus Propionibacterium, some of the strains being totally inactive (1, 11). P. granulo sum KP-45 whole cells and isolated cell walls protected mice against herpesvirus en cephalitis when administered a few days prior to the inoculation of viruses (21). Sev eral years ago we introduced another immunomodulating strain, namely Propionibac terium avidum KP-40 (PA) because of its better standardization possibilities. Both propionibacterial strains used have been shown to possess pronounced immunostim ulating potency with a low immunogenicity and no toxicity in mice (11, 17, 19). All experimental and clinical observation data collected for PA strongly suggest that PA may be a useful immunomodulating agent in clinical medicine (for review, see 3, 17). It has been the aim of the present investigations to study the prophylactic and ther apeutic potency of Propionibacterium avidum KP-40 (PA) in experimental herpesvirus encephalitis in mice, in comparison to earlier successful studies with the bacterial im munomodifier, P. granulosum KP-45 (30). Materials and Methods 1. Mice: NMRI mice used for the infection with herpes simplex viruses were obtained from the Rega Institute of Virology (Catholic University of Leuven, Belgium) by the cour tesy of Prof. E. de Clercq and grown at the Department of Virology in Warsaw, Poland. Mice were bred under standard conditions and used for experiments at the age of 6 weeks. 2. Herpes simplex virus (HSV-1): HSV-l (strain KOS) was propagated in primary rabbit kidney (PRK) cell cultures. Stock virus had about 10 6 TCIDso/mL (Tissue Culture Infectious Dose) and was stored at -70°C. For the infection of mice, the stock virus was diluted 1:2 in Parker medium; 0.02 mL of the virus suspension, corresponding to 104 TCID so per mouse, was diluted in 0.2 mL of Ringer's solution and injected intra peritoneally. This resulted in the development of herpes virus encephalitis (confirmed by a high titre of HSV-1 in brain tissues during earlier studies of this infection) with about 85% of control animals dying between the 7th and the 16th day after the virus inoculation. Mortality was noted daily, animals surviving 18 days after in fection without signs of illness were counted as survivors. 3. Propionibacterium avidum KP-40 (PA): PA (heat-killed, lyophilized bacteria) was ob tained from the Institute of Medical Microbiology and Hygiene, University of Cologne, Ger many. The bacteria were grown, harvested and prepared as described earlier (11, 17). Be fore use, all samples of PA were tested under standard conditions for their immunostimu la ting potency (17). For the treatment of mice, a stock suspension of PA in double-distilled water containing 5 mg/mL was prepared 4-6 h before use and stored at 4 0C. The stock suspension of PA was diluted 1:2 with Ringer's solution and 0.2 mL of the dilution containing 0.5 mg of PA was injected intra peritoneally once per mouse. 4. Acyclovir (ACL): Sodium 9([2-hydroxyethoxy]-methyl)-guanine in form of lyophilized Zovirax (Wellcome, Great Britain) for intravenous administration in 250 mg vials was used throughout the experiments. For the treatment of mice, a stock solution of ACL in 0.15 M phosphate buffer pH 7.4 containing 1 mg/mL was prepared and 0.2 of this solution was in-
Herpes Virus and Immunomodulation
447
jected intraperitoneally twice daily (at 8.00 a.m. and 8.00 p.m.) during 10 days after the in fection with HSV-l (corresponding to 15 mg/kg b.w.). 5. Poly I: C: Polyriboinosinic polyribocitidylic acid was purchased from Sigma Chemical Co. (St. Louis, MO, USA). For the treatment of mice, 0.2 mL of 200 ~g/mL solution of Po ly I: C in Ringer's medium (corresponding to 2.0 mg/kg b. w.) was injected intraperitoneal ly once daily for four days after the infection with HSV-1. 6. Experiments: Six-week-old male NMRI mice were randomly divided into 7 experi mental groups of 30 animals each (Table 1). All groups were infected intraperitoneally with 0.2 mL (104 TClD so ) of strain KOS HSV-l solution. Four days before the inoculation of vi ruses, on the day of infection or 2 days after HSV-l injection, the respective groups of mice (Table 1) were treated with Poly I: C, PA, ACL or a combination of these agents. Mortality was noted daily for 18 days following infection. 7. Statistical analysis: For analysis of the significance of differences in mortality between groups of animals, the chi-square test with Yates correction and a test for proportions (sta tistical calculator, CSS Statistica Win 4.5.) were used. In all cases, p < 0.05 was considered as significant.
Results and Discussion Inoculation of 104 TCID so of HSV-l resulted in death of 26 out of 30 (86.7%) of control mice (Table 1). The mortality started 7 days after infection, then grew rapidly and reached 50% of animals on the 10th day. Treatment with PA significantly lowered the mortality rate p < 0.01) (Table 1). Administration of ACL (15 mg/kg b.w.) on the day of HSV-l infection and on 10 consecutive days lowered the mortality rate to 36.7% (p < 0.01), but still about 113 of HSV-1 infected mice died within 18 days. The survi val of HSV-1 infections could be considerably increased by a combined treatment with ACL and PA. Administration of ACL for 10 days after HSV-1 infection combined with a single injection of PA on the day of virus inoculation resulted in a survival rate of 28 out of 30 mice (Table 1). This combination of treatment significantly enhanced the sur vival rate when compared not only to controls but also to the Poly I: C-treated group. The model of experimental HSV-l encephalitis in mice allows a valid testing of pro phylactic and therapeutic modalities. The course of experimental HSV-l infections in
Table 1. Survival rates of NMRI mice and results of different treatment schedules Group of mice (N
= 30)
Controls (untreated) Poly I:C 2 mg/kg (with HSV) PA 25 mg/kg, 4 days before HSV PA 25 mg/kg with HSV PA 25 mg/kg, 2 days after HSV ACL 15 mg/kg with HSV ACL + PA with HSV
18-day survival after HSV-l infection Number of mice surviving
Survival rate
4 18 22 21 17 19 28
0.133 0.60* 0.733* 0.70* 0.566* 0.633* 0.933*@
* p < 0.01 - compared to controls (untreated). @ p< 0.01 - compared to Poly I: C-treated group. PA = Propionibacterium avidum KP-40; ACL =Acyclovir.
448
M. Kobus et a1.
mice depends on the strain of animals, their age, the strain of viruses and the way of inoculation (6). In general, C 3H and BALB/c inbred mouse strains and most of their hybrids are considered as being relatively resistant to herpesviruses, while NMRI and Swiss mice belong to sensitive strains (18). NMRI mice were often challenged for ex perimental investigations of HSV-1 infections and testing of therapeutic modalitis (6, 21), however, in most of the earlier studies the course of experimental infections was very fast with nearly all animals dying within a few days after virus inoculation. For example, in our earlier study on the protection and therapy of experimental HSV-1 in fections in mice with Propionibacterium granulosum KP-45 (21), the intranasal infec tion of 12-day-old NMRI mice resulted in a mortality of 50% of control animals dur ing 4-5 days and death of 95% of mice within 9 days after infection. This rapid de velopment of disease left no time for modulations of the immune responses when pro pionibacteria were administered 3 days after the inoculation of viruses and, therefore, the mortality was only slightly lowered in this group. The model applied in the present study used older mice (6 weeks instead of 12 days), a different way of inoculation (intraperitoneal instead of intranasal) and a lower infective dose (10 4 TCID so ). These modalities resulted in a slower development of the disease, with all animals surviving the first 6 days after infection. Mortality started 7 days after virus inoculation and rap idly grew up to 16 days after infection, when about 85% of control animals had died. The bacterial immunomodifier PA tested in the present study exhibited prophylactic and therapeutic potency. PA administered either 4 days before or 2 days after the inoc ulation of viruses lowered the mortality of HSV-1-infected mice from 87% to 27-44% and prolonged survival of the animals. In the control group, mortality started 7 days af ter infection, while in PA-treated groups the first dying animals appeared a few days lat er (8-10 days after inoculation of viruses). In the present study, the best protection against mortality from herpes virus infections was obtained by the treatment of HSV-1infected mice with a combination of antiviral ACL and immunomodulation with PA. The treatment started on the day of virus inoculation and lasted during the first days af ter the infection, being therefore considered as a therapeutic modality with some clini cal relevance. The applied therapy protocol lowered the mortality rate from 87% in the controls to 7%, being significantly lower (p < 0.01) when compared with mortality rates of animals treated with ACL, PA or Poly I:C alone. ACL is a herpes-specific antiviral substance which inhibits the multiplication of virus particles and therefore limits the spread of the infection (16). In local infections with HSV (e. g. skin infections, keratitis), ACL totally inhibits virus replication and allows a cure of the infection (15). However, in general infections of sensitive hosts with large infective doses of viruses, only a par tial protection is possible (2). In the present model, the use of ACL at a dose of 15 mg/kg b. w. for 10 days after infection resulted in lowering the mortality from 87% to 37%. References 1. Adlam, C. and M. T. Scott: Lymphoreticular stimulatory properties of Corynebacterium parvum and related bacteria. J. Med. Microbiol. 6 (1973) 262-274 2. Balfour, H. H., C. Benson, J. Braun, B. Cassen, A. Erice, A. Friedman-Kien, T. Klein, B. Polsky, et a1.: Management of acyclovir-resistant herpes simplex and varicella zoster virus infections. J. Acquired Immun. Def. Syndr. 7 (1994) 254-260 3. Beuth, J., H. L. Ko, K. M. Peters, B. Bornhofen, and C. Pulverer: Behaviour of lympho cyte subsets in reponse to immunotherapy with Propionibacterium avidum KP-40 in can cer patients. Zb1. Bakt. 273 (1990) 386-390
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w.,
Prof. Dr. Dr. h. c. G. Pulverer, Inst. f. Med. Mikrobiologie und Hygiene der Universitat, Goldenfelsstrage 19-21, D-50935 Kaln, Germany