Antiherpes activity of the immunomodulator OK-432, a streptococcal preparation, in immunosuppressed mice

Antiviral Research, 10 (1988) 299-304

299

Elsevier AVR00324

Short Communication

Antiherpes activity of the immunomodulator OK432, a streptococcal preparation, in immunosuppressed mice Satoru Ikeda 1, Kazutoshi Sai 1, Chiaki Nishimura 1 and Akihiro Yamamoto 2

1School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108; 2Chugai Pharmaceutical Co. Ltd., Toshima-ku Tokyo 171, Japan

(Received 21 June 1988; accepted 18 October 1988)

Summary The antiviral activity of OK-432, an antitumor agent originating from Streptococcal preparations, against herpes simplex virus type 2 (HSV-2) was investigated in mice immunosuppressed by cyclophosphamide (CY). Intraperitoneal administration of OK-432 to mice 1 day after treatment with 200 mg CY/kg prevented death due to HSV-2 encephalitis in a dose-dependent manner. When the immunosuppressed mice were given OK-432 prior to HSV-2 infection, both by the intraperitoneal route, virus growth in the peritoneal cavity was significantly suppressed. Following with OK-432, the number of macrophages in immunosuppressed mice was increased to a significantly greater extent than the numbers of lymphocytes and polymorphonuclear leukocytes. The intrinsic antiviral activity of macrophages against HSV-2 as well as the natural killer (NK) activity against YAC-1 target cells was significantly enhanced by OK-432 in immunosuppressed mice. OK-432; Immunomodulator; Herpes simplex virus; Immunosuppressed mice

During a primary HSV infection, the virus migrates from the infected area to a ganglion where it establishes a latent infection. The latent HSV has a tendency to Correspondence to: Dr. C. Nishimura, Department of Virology and Immunology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo 108, Japan.

0166-3542/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

300 be activated especially during immunosuppression, occasionally resulting in very severe disease. On rare occasions, HSV infections may be generalized in immunodeficient patients (Nahmias et al., 1976; Wheeler, 1975). HSV infections are also thought to be oncogenic, particularly in the development of cervical carcinoma (Rapp, 1973). Among a number of remedies tested for this recrudescent disease, acyclovir and foscarnet have been found to be therapeutically active (Alenius et al., 1982; Field et al., 1979). The present study was aimed at investigating the efficacy of an immunomodulator against HSV infection and establishing its mode of action. OK-432 is a streptococcal preparation first developed in Japan and widely used as an antitumor agent (Hojo and Hashimoto, 1981; Kondo et al., 1975; Sakurai et al., 1972; Uchida and Hoshino, 1980). However, conclusive information as to how OK-432 enhances host-defense mechanisms against tumors is lacking. Possible mechanisms include the induction of interferon (IFN) (Saito et al., 1982) and tumor necrosis factor (TNF) (Yamamoto et al., 1985), and augmentation of NK activity (Oshimi et al., 1980; Uchida and Micksche, 1983). Since tumor patients are usually compromised in their immune system and vulnerable to virus infection, we investigated whether the immunomodulator OK-432 could restore the host-immune system and prevent HSV-2 spreading in immunosuppressed mice. Female ddY mice bred for one week in a specific pathogen-free environment were used for experiments at 5 weeks of age. The UW strain of herpes simplex virus type 2 (HSV-2) grown in HEL (human embryonic lung) cells was stored at -80°C and used for the animal experiments. OK-432 (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan) and CY (Shionogi Co. Ltd., Osaka, Japan) were dissolved in sterile physiological saline immediately before use. Groups of 10 mice were injected intraperitoneally (i.p.) with 200 mg CY/kg 4 days before virus infection. OK-432 diluted to various concentrations was then administered i.p. to CY-suppressed mice at various time intervals. After i.p. infection with 1 x 103 plaque-forming units (PFU) of HSV-2, the time of death was recorded daily for 14 days, and the percentages of surviving mice in experimental and control groups were compared statistically by the chi-square test. Peritoneal cells were obtained by lavage of the peritoneal cavity with Eagle's minimum essential medium (EMEM) according to the routine method described in a previous paper (Ikeda et al., 1985). The ratios of macrophages, polymorphonuclear leukocytes (PMN), and lymphocytes to the total peritoneal cell number were determined by microscopic observation after Giemsa staining. For assay of intrinsic antiviral activity of macrophages, peritoneal cells were harvested from normal or CY-treated immunosuppressed mice which had been given i.p. 100 p~g OK-432 3 days previously. The activity against HSV-2 was assessed by determining virus growth in macrophages (Ikeda et al., 1985). Macrophages prepared at a cell concentration of 5 x 105/well were cultivated for 2 h at 37°C in a 24-well culture tray and infected with HSV-2 at a multiplicity of infection (MOI) of 0.1. After adsorption for 1 h, unadsorbed viruses were washed out twice with EMEM. The RPMI-1640 medium containing 10% fetal calf serum was added to the cells. After cultivation for 24 h at 37°C, the cells were harvested with a rubber policeman and disrupted by two freeze-thawing cycles. The virus ti-

301 ters in the cell h o m o g e n a t e s were determined by a plaque assay on 3T3 monolayer cells. For assay of N K cell activity, the peritoneal cells were obtained from normal or CY-suppressed mice which were given i.p. 100 ~g OK-432 3 days before the assay. N K activity was assessed by determining the radioactivity released from 5~Crlabeled YAC-1 target cells (Ikeda et al., 1988). Briefly, the peritoneal cells were incubated for 4 h at 37°C with 51Cr-labeled target cells in an effector:target ratio of 30:1. The cell mixtures were prepared in triplicate and the radioactivity released into the supernatant was counted in an a u t o g a m m a scintillation spectrometer. The specific release of radioactivity was calculated according to the following formula: Specific lysis ( % ) = {(cpm of tested groups - cpm of the spontaneous release)/(cpm of the complete release - cpm of the spontaneous release)} x 100. The N K cell activity detected in the nonadherent fraction could be neutralized by treatment with anti-asialo GM~ monoclonal antibody (Wako Chemicals, Co. Ltd., Tokyo). When CY was administered i.p. to mice at a dose of 200 mg/kg 4 days before i.p. challenge with 103 P F U of HSV-2, mortality of CY-suppressed mice markedly increased in comparison with that of untreated mice. The mortality of immunosuppressed mice was reduced by preliminary administration of OK-432 between days 1 and 3 before HSV-2 challenge. As seen in Fig. 1, the protective activity of OK-432 was dose-dependent and most effective when 100 ~g OK-432 was administered i.p. to CY-suppressed mice 3 days before HSV-2 infection. Growth of HSV2 in the peritoneal cavity of CY-suppressed mice was enhanced 4-fold c o m p a r e d with that in untreated mice. Viral growth in the peritoneal cavity of CY-suppressed mice was markedly reduced following i.p. administration of 100 p,g OK432 3 days before HSV-2 infection (data not shown). 100

75

50

25

0

0

i

I

I

5

10

14

Days after infection

Fig. 1. Effect of OK-432 on the induction of resistance against HSV-2 in immunosuppressed mice. Groups of 10 mice were treated i.p. with 200 mg CY/kg 4 days previously and given i.p. different doses of OK-432 3 days before i.p. challenge with 1 x 1 0 3 PFU of HSV-2. o, control; e, CY-suppressed; A, CY-suppressed and treated with OK-432 (100 ~zg); A, CY-suppressed and treated with OK-432 (10 i.tg); E3, CY-suppressed and treated with OK-432 (1 t.tg). *P < 0.005, **P < 0.01, as compared with CYsuppressed mice (×2 test).

302 TABLE 1 Effect of OK-432 on the recovery of peritoneal cells in immunosuppressed micea Treatment Non-treatment CY(200 mg/kg, at -4d) CY(200 mg/kg, at -4d)+ OK-432(100 ~g, at -3d)

Cell number (x 106/mouse) Total cells

Macrophages

PMNs

Lymphocytes

5.62 + 0.69 1.91 -+ 0.23

2.92 --_ 0.36 0.82 --_ 0.10

0.06 _+ 0.01 0.10 +- 0.01

2.14 +- 0.25 0.96 +- 0.12

3.39 -+ 0.16b

1.90 ~ ().09h

0.07 _+ 0.01

1.36 -+ 0.07c

aPeritoneal cells were harvested from mice treated i.p. with CY (200 mg/kg, at -4d) and OK-432 (100 p.g, at -3d). The number of PC was counted using a hemocytometer, and cell population of the PC was determined microscopically by Giemsa staining. bp <~ 0.001 cp < 0.01, as compared with CY-suppressed mice (Student t-test). T o t a l cell n u m b e r a n d cell p o p u l a t i o n in the p e r i t o n e a l cavity of n o r m a l a n d imm u n o s u p p r e s s e d mice w e r e e x a m i n e d as s h o w n in T a b l e 1. T h e total n u m b e r of p e r i t o n e a l cells ( P C ) in i m m u n o s u p p r e s s e d mice was r e d u c e d 3-fold as c o m p a r e d to u n t r e a t e d mice. U p o n a d m i n i s t r a t i o n o f O K - 4 3 2 , the total P C n u m b e r inc r e a s e d a b o u t 2-fold in i m m u n o s u p p r e s s e d mice. T h e i n c r e a s e in m a c r o p h a g e n u m b e r was g r e a t e r t h a n the i n c r e a s e in the n u m b e r of l y m p h o c y t e s a n d P M N . A n t i v i r a l activity of p e r i t o n e a l m a c r o p h a g e s was assessed ex vivo by t i t r a t i o n of viral g r o w t h in t h e m a c r o p h a g e s of i m m u n o s u p p r e s s e d mice, A s s h o w n in T a b l e 2, H S V - 2 r e p l i c a t e d m o r e r e a d i l y in m a c r o p h a g e s f r o m C Y - s u p p r e s s e d mice t h a n in m a c r o p h a g e s from u n t r e a t e d mice. H o w e v e r , when i m m u n o s u p p r e s s e d mice had b e e n t r e a t e d with 100 Cg O K - 4 3 2 3 d a y s b e f o r e H S V - 2 infection, viral r e p l i c a t i o n was s u p p r e s s e d . V i r u s titers in m a c r o p h a g e s f r o m i m m u n o s u p p r e s s e d mice t r e a t e d with O K - 4 3 2 w e r e l o w e r t h a n virus titers in m a c r o p h a g e s f r o m i m m u n o s u p p r e s s e d mice which h a d n o t b e e n t r e a t e d with O K - 4 3 2 , i n d i c a t i n g that OK-432 r e s t o r e d a n t i v i r a l activity of m a c r o p h a g e s d e r i v e d f r o m C Y - i m m u n o s u p p r e s s e d mice. T h e N K activities in p e r i t o n e a l cells of t h r e e e x p e r i m e n t a l g r o u p s ( n o r m a l , CYs u p p r e s s e d , a n d C Y - s u p p r e s s e d O K - 4 3 2 - t r e a t e d mice) w e r e assessed by d e t e r m i n ing radioactivity r e l e a s e d from 51Cr-labeled Y A C - 1 target cells. The peritoneal cells w e r e o b t a i n e d f r o m n o r m a l or C Y - s u p p r e s s e d mice which h a d b e e n given i.p. 100 Ixg O K - 4 3 2 3 d a y s p r e v i o u s l y . A s s h o w n in T a b l e 3, N K activity was c o m p l e t e l y TABLE 2 Effect of OK-432 on the intrinsic antiviral activity of macrophages of immunosuppressed mic& Macrophage source

Virus titer (PFU/ml)

Ratio

Non-treatment CY (200 mg/kg, at -4d) CY (200 mg/kg, at -4d) + OK-432 (100 ~g, at -3d)

(5.3 -+ 2.1) × 102 (1.3 +- 0.6) × 103

1.0 2.5 (1.0)

(1.6 +- 0.6) × 102b

0.3 (0.1)

aThe antiviral activity of peritoneal macrophages obtained from normal or CY-treated mice given i,p. 100 p-g OK-432 was assessed by determining the virus growth in macrophages. bp < 0.001, as compared with CY-suppressed mice.

303 TABLE 3 Effect of OK-432 on NK cell activity in immunosuppressed micea NK cell source

Specific lysis (%)

Non-treatment CY (200 mg/kg, at -4d) CY (200 mg/kg, at -4d) + OK-432 (100 ~g, at -3d)

2.0 + 0.8 - 0.9 _+ 0.8 11.6 -+ 0.9 b

~The NK cell activity of peritoneal cells obtained from normal or CY-suppressed or CY-suppressed OK432-treated mice was assessed by determining the radioactivity released from 5~Cr-labeled YAC-1 cells, as described in the text. ~P < 0.001, as compared with CY-suppressed mice. a b o l i s h e d in p e r i t o n e a l cells o f C Y - s u p p r e s s e d mice. H o w e v e r , the activity rec o v e r e d if the C Y - i m m u n o s u p p r e s s e d mice h a d b e e n t r e a t e d with O K - 4 3 2 . T h e I F N titer o b t a i n e d in t h e m o u s e p e r i t o n e a l fluid 24 h after i.p. i n j e c t i o n of 1 x 107 P F U of H S V - 2 to n o r m a l a n d C Y - s u p p r e s s e d mice was 190 a n d 135 U/0.1 ml, respectively. A d m i n i s t r a t i o n o f O K - 4 3 2 by itself did not result in the p r o d u c t i o n o f peritoneal IFN (data not shown). B u h l e s a n d Shifrine (1977) r e p o r t e d that a d m i n i s t r a t i o n of C Y t o mice r e s u l t e d in m a r k e d reduction in n u m b e r s of various cell types including PMNs, b l o o d m o n o cytes and s u b p o p u l a t i o n s of lymphocytes, and that injection of Mycobacterium bovis B C G o r F r e u n d c o m p l e t e a d j u v a n t into C Y - s u p p r e s s e d mice i n d u c e d r a p i d rest o r a t i o n o f cell n u m b e r o f p e r i p h e r a l b l o o d g r a n u l o c y t e s . T h e p r o t e c t i v e effect o f O K - 4 3 2 a g a i n s t H S V - 2 i n f e c t i o n in C Y - s u p p r e s s e d mice is d e p e n d e n t on effective r e c r u i t m e n t o f b l o o d m o n o c y t e s into the p e r i t o n e a l cavity a n d s t i m u l a t i o n of t h e i r f u n c t i o n a l m a t u r a t i o n in situ. A d m i n i s t r a t i o n o f O K - 4 3 2 e n h a n c e d the intrinsic antiviral activity of m a c r o p h a g e s a n d N K activity in i m m u n o s u p p r e s s e d m i c e , suggesting t h a t t h e a c t i v a t i o n o f m a c r o p h a g e s a n d N K cells p l a y s an i m p o r t a n t role in the c l e a r a n c e o f H S V - 2 a n d H S V - 2 - i n f e c t e d cells in i m m u n o s u p p r e s s e d mice. In s u m m a r y , o u r results i n d i c a t e t h a t O K - 4 3 2 a u g m e n t s non-specific resistance against viral i n f e c t i o n in i m m u n o c o m p r o m i s e d mice. Since t h e r e was a r e v e r s e d relationship b e t w e e n m a c r o p h a g e / N K cell functions and HSV-2 replication in mouse p e r i t o n e a l cavity, O K - 4 3 2 a p p e a r s to be effective in s u p p r e s s i n g H S V - 2 infection in i m m u n o c o m p r o m i s e d hosts.

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