Effects on Friend Disease of Double-stranded RNA of Fungal Origin

Transcription

1 J. gen. ViroL (I97~), io, I55-I Printed in Great Br#ain Effects on Friend Disease of Double-stranded RNA of Fungal Origin By D. J. F. PILCH AND D. N. PLANTEROSE Beecham Research Laboratories, Brockham Park, Betchworth, Surrey (Accepted t2 October I97O ) SUMMARY This paper reports the effects of treatment with double-stranded RNA of Friend disease, a virus-induced murine leukaemia. The salient feature of the disease is a progressive increase in spleen size; death normally results from rupture of the spleen. It was found that the effect of treatment with double-stranded RNA was closely related to the time of treatment relative to infection. Treatment before or in the early stages of infection increased the severity of the disease, but treatment 5 days after infection led to a profound reduction in the severity of the disease, judged by a pronounced reduction or abolition of splenomegaly. Histological examination of this reversal of splenomegaly showed reduction of the Friend cell infiltration and a return to a more normal spleen architecture. Mice in which remissions have been produced have remained free of evidence of splenomegaly for several weeks. The nature of this therapeutic effect is unknown. INTRODUCTION It has been reported that Statolon, a product of the mould Penicillium stoloniferum, was effective in inhibiting Friend virus-induced leukaemia in mice if given before infection (Wheelock, I967). It has also been reported that one injection of Statolon 3 days after infection can produce long term clinical remissions in DBA2 mice infected with Friend virus (Wheelock, Caroline & Moore, I969). The antiviral and interferon-inducing activity of Statolon, originally thought to be a polysaccharide, is now known to be due to double-stranded RNA found in virus-like particles present in the moulds (Banks et al. I968, Kleinschmidt et al. I968). Some years ago, in co-operation with Professor Sir Ernst Chain, we undertook a study on the biological and chemical properties of double-stranded RNA obtained from such moulds. The first paper described the effect of these interferon inducers on acute virus infections (Planterose et al. I97o). With the availability of highly purified double-stranded RNA it was also of interest to investigate the effect of this on Friend disease, an example cf a non-acute virus infection. This paper reports on experiments to determine the effect of highly purified double-stranded RNA on Friend disease with special reference to the relationship between response and time of treatment relative to infection. Recent studies on the effect of the synthetic polynucleotide poly I:C on Friend disease (Larson et al. I969a) have shown that there is a complex relationship between the effect on Friend virus and the time of poly I: C administration. Although there is a profound difference in the biological activity of different synthetic polynucleotides (e.g. between poly I: C and poly A: U), it is believed that the biological actions of poly I: C and naturally produced double-stranded RNAs are similar (Nemes et al. I969). The present results give in more detail the time of treatment after infection for antitumour effect.

2 I56 D. J. F. PILCH AND D. N. PLANTEROSE METHODS Friend virus was obtained from Dr M. Salaman, Department of Cancer Research, London Hospital Medical School, in the form of infected female Balb/C mice. The virus was passaged using the plasma of infected mice. Blood was collected by cardiac puncture and a 20 solution was made in phosphate-buffered saline containing 20 units heparin/ml. The blood cells were removed by centrifugation at 2000 g for 20 rain. and the supernatant fluid passed through an HP/EK filter (Carlson-Ford, pore size 45o nm.) and stored in ampoules at - 7o. A standard stock of plasma virus was used in all experiments. Mice. All experiments were carried out in male CD I mice of ~8 to 22 g., obtained from Charles Rivers, France. Double-stranded RNA was produced at Beecham Research Laboratories from a penicillium culture which contained a large amount of double-stranded RNA. The doublestranded RNA, when highly purified, was free from protein and DNA and had a sedimentation coefficient of approximately I2S. It exhibited marked hyperchromicity and a melting profile when heated and was relatively resistant to bovine pancreatic ribonuclease (Koch- Light Laboratories). Assay of infection in mice. The progress of the disease was determined by comparison 14 days after infection of the spleen weights of the treated group with those of the control group. The ratio of spleen weight to body weight was also determined for each mouse. The results were subjected to standard statistical analysis. RESULTS Effect of a single dose of double-stranded RNA on splenomegaly induced by Friend virus Double-stranded RNA was injected intraperitoneally into CD I mice at ~oo #g./mouse at various times. The results show (Table I) that, depending on the time of administration relative to infection at day o, the effect could be enchancement, no effect, or inhibition of splenomegaly. Enhancement of splenomegaly occurred when double-stranded RNA was injected from 5 days before, or up to 3 days after infection. The mechanism of enhancement of Friend virus-induced splenomegaly is probably related to the immunosuppressive activity of the double-stranded RNA, since it is known that immunosuppressive agents enhance splenomegaly. Thus immunosuppression by antilymphocyte serum of Rauscher virus produces a similar increase in splenomegaly (Hirsh & Murphy, ~968). In separate experiments the immunosuppressive action of the double-stranded RNA used here has been demonstrated (to be published). Significant inhibition of splenomegaly occurred if double-stranded RNA was injected from day 7 up to day 11 after infection. This regression was rapid, as shown in a subsequent experiment in which inhibition of splenomegaly occurred at day I4 when ~oo #g./mouse of double-stranded RNA were injected on day ~3 (Fig. i). Fig. ~ also shows the time course of splenomegaly in untreated mice infected at day o, as determined by sampling groups of lo mice on the days shown. The inhibition of splenomegaly produced by dosing later in infection has been interpreted as being mediated by interferon (Gresser et al. I967). On the other hand, a more specific antiviral or antitumour effect, or a stimulation of the immune response, may be involved. Further groups of mice dosed at day ~o remained free of evidence of splenomegaly for several weeks, but by Io weeks after infection 70 ~ of these mice had significant

3 Effect of double-stranded RNA on Friend disease 157 Table J. Comparison of spleen weights at 14 days of mice injected intraperitoneally with a single dose of IOO #g. of double-stranded RNA Day of treatment relative to infection Log. dilution of Friend virus --7 IO z -- 5 IO I0-1 --I I0 1 0 I0-1 +I IO z +5 I IO-1 +9 Io ~ + I I I0-1 Untreated IO 1 Untreated to 2 Untreated lo Untreated Uninfected Spleen wt. (mg.) Spleen wt (mg.):body wt (g.) No. of c ~ ~ ( " mice/ Standard Significance Standard Significance group Mean deviation level (%) Mean deviation level* (%) NS 17"3 5"8 NS io o.i 27'2 i2-o 1.o I I'0 I9"I 6'6 I'O IO o.i o-i IO O'I 22"7 9'7 2'0 1o I.O 23"9 8.6 O'I IO o o io 336 I94 20'o I2" IO "3 2.o o.i io i-o o.i I "I 7" 'I "4 5"9 -- lo I.O 8" I I'0 25"1 1"9 I'0 I I 4"I 0'9 0"I * Significance level with respect to untreated mice infected with 1o -1 dilution of virus; NS, not significant ~o 1000 t I I 1 I I I 1 1 I I I I I o 600 \ / "'",.~ '200 ~. 'A... _,... ;i -;-_ ;---- ;:: ~ i I I i i = i I I I I I I I Days after infection Fig. I. Time course of Friend virus-induced splenomegaly in treated and untreated mice. A batch of mice were infected at day o. Groups of these mice were treated with a single dose of double-stranded RNA (IOO #g./mouse intraperitoneally) at day IO,... ; at day I3, --- 0; left untreated, R--I. The course of splenomegaly in these groups was followed by sampling groups of ten mice at the times shown. The mean spleen weights of mice uninfected and untreated are shown for comparison ( ---O).

4 I58 D.J.F. PILCH AND D. N. PLANTEROSE splenomegaly. This induction of a latent state of the disease is similar to that of Statoloninduced remissons (Wheelock et al. I969 ). No effect on splenomegaly occurred if mice were treated on day + 5 (Table ~). This may reflect interplay of the immunosuppressive and antileukaemic action of double-stranded RNA, or a non-reactive state. No effect was observed on the spleen size of uninfected mice following injection of doublestranded RNA. Treatment Table 2. Effect on spleen weight at day I4 of mice dosed intraperitoneally with 5o #g. of double-stranded RIgA on alternate days for various periods Log. Spleen wt (rag.) Spleen wt (mg.):body wt (g.) period dilution No. of r ' ~,- ~" relative to of Friend mice/ Standard Significance Standard Significance infection virus group Mean deviation level (%) Mean deviation level* (%) Days - 7 lo -1 lo o" I 34"0 11"o o'i to -- I Days + I IO -1 IO o.o 19"3 7"5 5'0 to -t-7 Days +7 IO -1 lo Po 8'6 2.I 2'o to +ii Days + i io -1 io 41I 145 NS 14"8 5"5 NS to +1i Untreated IO "4 5"9 -- Untreated lo -2 Io I.o 8"4 2.2 i.o Untreated 1o lo I 1.o 25'1 1"9 1.o Untreated Uninfected Io I5o 25 o'i 4"I 0"9 o.i * Significance level with respect to untreated mice infected with lo -1 dilution of virus; NS, not significant. Effect of multiple dosing Further groups of mice were inoculated intraperitoneally with double-stranded RNA at 50 #g./mouse on alternate days (Table 2) and the effect on splenomegaly at day 14 compared with that for the infected controls. As with single dose administrations, three patterns occurred: 0) enhancement of splenomegaly at day I4 for doses given before infection (days - 7 to - I) or in the first week of infection (days + I to + 7); (2) reduction in spleen weight at I4 days for doses given in the 2nd week of infection (days +7 to + II); (3) no significant effect on splenomegaly at day I4 for doses given on days + I to -t-i I. These results may be interpreted as in the single dose experiments. Histological examination of spleens Spleens of uninfected mice show a discrete arrangement of red and white pulp (Fig. za). In Friend virus-infected spleens the normal architecture is disrupted by massive infiltration of sheets of'friend cells' into the red pulp (Fig. zb). Spleens from infected mice ultimately become necrotic and haemorrhagic (Fig. 3a). Histological examination of the spleens of mice in which reduction of splenomegaly has resulted from treatment with double-stranded RNA show a return to more normal spleen structure (Fig. 3b). DISCUSSION It is apparent that the administration of double-stranded RNA can have profound effects on the course of Friend virus-induced splenomegaly. The effects are various and related to the time of injection of double-stranded RNA relative to infection.

5 Effect of double-stranded RNA on Friend disease Fig. z. (a) Normal spleen showing arrangement of red and white pulp (arrowed). ~75. (b) Infected spleen, weight 620 mg., showing infiltration of red pulp by sheets o f ' Friend cells' (arrowed) I59

6 16o D. J. F. P I L C H A N D D. N. P L A N T E R O S E Fig. 3. (a) Necrosis and haemorrhage in a Friend virus-infected spleen weighing 2-2 g. x I75. (b) Spleen of Friend virus-infected mouse treated with double-stranded R N A at day I I showing regression of Friend cell foci and a return to the normal architecture of red and white pulp (arrowed). x I75.

7 Effect of double-stranded RNA on Friend disease The period during which the infection was susceptible to inhibition by double-stranded RNA appeared to correspond with the phase of the disease when rapid proliferation of Friend cells was causing the spleens to increase in size. The effect therefore appeared to be on proliferating cells or tissue. So it is of interest to note the disappearance of the Friend cell in treated spleens. The remission induced following one injection of double-stranded RNA had not relapsed 6 weeks after treatment, suggesting that the reduction in spleen weight was more than a temporary reduction in the number of Friend cells. The mechanism by which the reversal of splenomegaly is produced is uncertain. Doublestranded RNA is a potent inducer of serum interferon which could be acting as an antiviral agent on Friend virus multiplication. Interferon could also be acting on the Friend cells themselves in a similar manner to its antitumour action described by Gresser (Fontaine- Brouty-Boy6 et al. I969; Gresser & Bourali, 1969). On the other hand, the antitumour action may not be related to the interferon-inducing ability of double-stranded RNA. Poly I: C is known to exert an antitumour action against a variety of tumours such as a reticulum cell sarcoma in mice and adenovirus I2 in newborn hamsters (Levy, Law & Rabson, I969; Bart & Kopf, I969; Larson, Clark & Hilleman, I969b). Most probably the synthetic and naturally occurring double-stranded RNAs have a common mode of action. The rapidity of the regression of infected spleens would tend to rule out the involvement of antibodies and delayed hypersensitivity immune mechanisms in the response. Certain aspects of work with poly I: C have been criticized in that the absence of endotoxin contamination had not been established when assaying biological and toxicological activity (Hilleman, I97o). The activity against Friend disease of our material has been shown to be due to double-stranded RNA which is Schwartzman negative. 16 I REFERENCES BANKS, G. T., BUCK, K. W., CHAIN, E. B., HIMMELWEIT, F., MARKS, J. E., TYLER, J. M., HOLLINGS, M., LAST, F. T. & STONE, O. M. (I968). Viruses in fungi and interferon stimulation. Nature, London 218, 542. BART, R. S. & KOPF A. W. (I969). Inhibition of the growth of murine malignant melanoma with synthetic double-stranded ribonucleic acid. Nature, London 224, 372. FONTAINE-BROUTY-BOYi~, D., GRESSER, I., MACIEIRA-COELHO, A., ROSENFELD, C. & THOMAS, m. (t969). Inhibition in vitro de la multiplication des cellules leuc6miques murines L I2IO par des pr6parations d'interf6ron. Compte rendu hebdomadaire des sdances de l'acaddmie des Sciences, Paris 269, 4o6. GRESSER, t. & BOURALI, C. (1969)- Exogenous interferon and inducers of interferon in the treatment of Balb/C Mice inoculated with RC I9 tumour cells. Nature, London 223, 844. GRESSER, I., FALCOFF, R., FONTAINE-BROUTY-BOYE, D., ZAJDELA, F., COPPEY, J. & FALCOFF, E. (1967). Interferon and murineleukemia. IV. Further studies on the efficacy of interferon preparations administered after inoculation of Friend virus. Proceedings of the Society for Experimental Biology and Medicine x26, (3) 791. HILLEMAN, i. R. (1970). Prospects for the use of double-stranded ribonucleic acid (Poly I: C) inducers in Man. Journal of Infectious Diseases 121, 196. mrsch, M. S. & MURPHY, E. A. (1968). Effects of anti-lymphoid sera on viral infections. Lancet (7558) ii, 37. KLEINSCHMIDT, W. J., ELLIS, L. F., VAN FRANK, R. M. & MURPHY, E. B. 0968). Interferon stimulation by a doublestranded RNA of a mycophage in Statolon preparations. Nature, London 22o, I67. LARSON, V. M., CLARK, W. R., DAGLE, G. E. & mlleman, N. R. (I969a). Influence of synthetic double-stranded ribonucleic acid, Poly I:C, on Friend leukemia in mice. Proceedings of the Society for Experimental Biology and Medicine I32, 6o2. LARSON, V. i., CLARK, W. R. & HILLEMAN, M. R. (I969b). Influence of synthetic (PolyI:C)and viral doublestranded ribonucleic acids on adenovirus i2 oncogenesis in hamsters. Proceedings of the Society for Experimental Biology and Medicine 13 r, loo2. LEVY, H. B., LAW, W. L. & RABSON, A. S. 0969). Inhibition of tumor growth by Polyinosinic-polycytidylic Acid. Proceedings of the National Academy of Sciences t~f the United States t~f America 62, 357. NEMES, M. M., TYTELL, A. A., LAMPSON, G. P., FIELD, A. K. & HILLEMAN, M. R. (1969). Inducers of interferon and host resistance. VII. Antiviral efficacy of double-stranded RNA of natural origin. Proceedings of the Society for Experimental Biology and Medicine I32, 784. II VIR IO

8 I62 D.J.F. PILCH AND D. N. PLANTEROSE VLANTEROSE, D. N., BIRCH, V. J., PILCH, n. J. F. & SHARJ?E, Z. J. (1970). Antiviral activity of the double-stranded RNA and virus-like particles from Penicillium stoloniferum, Nature, London 227, 5o4. WI-mELOC, Z. V. (1967). Effect of Statolon on Friend virus leukemia in mice. Proceedings of the Society for Experimental Biology and Medicine I24, 855. WHEELOCK, Z. V., CAROLINE, N. L. & MOORE, R, D, 0969)- Suppression of established Friend virus leukemia by Statolon. Journal of Virology 4, I. (Received 2I April 197o)