JOURNAL OF INTERFERON RESEARCH 10:293-298 (1990)Mary Ann Liebert, Inc., Publishers SHORT COMMUNICATION

Comparative Therapeutic Efficacy of RecombinantInterferons-a, -ß, and -7 Against Alphatogavirus,Bunyavirus, Flavivirus, and Herpesvirus Infections

ANGELO J. PINTO,1 PAGE S. MORAHAN,1 MARGO BRINTON,2,3DENEEN STEWART,' and EDITH GAVIN2

ABSTRACT

Recombinant (r) preparations of interferons (IFN)-a, -ß, and -y were shown to protect miceagainst experimental virus infections with herpes simplex virus type 2 (HSV-2), and withthree RNA-containing viruses from different families: Banzi, a flavivirus; Semliki Forestvirus (SFV), an alphatogavirus; and Caraparu, a bunyavirus. The antiviral effects of thethree different types of IFN were different with each virus. HSV-2 was the most sensitivevirus, followed by SFV. Against Banzi virus, IFN-7 was only effective when given bothbefore and after infection. Against Caraparu virus, only IFN-7 had a significant effect.These results suggest that IFN therapy might be valuable in human infections with theseviruses, but that the correct choice of IFN and dose regimen is likely to be important.

Infections caused by RNA viruses belonging to the Alphatogaviridae, Bunyaviridae, and Flaviviridaefamilies are an important health problem worldwide.'" In a number of studies these viruses have been

found sensitive to inhibition by interferon (IFN) or inducers;'2-9' however, there has been no systematiccomparison of the relative efficacy in vivo of the three types of IFN, IFN-a,

-

ß, and -y, against representativeviruses from these families. Such a study is reported here: mice were infected with Banzi, a flavivirus;Semliki Forest virus (SFV), an alphatogavirus; or Caraparu, a bunyavirus, and treated with one of three typesof recombinant IFN before, or after, or both before and after infection. For comparison, mice infected withherpes simplex virus type 2 (HSV-2) were included in the study.

Virus-free, barrier raised, 5-week-old female B6C3F, mice purchased from Taconic farms (Germantown,NY) were shipped and held under appropriate conditions of isolation. To ensure that no intercurrent viralinfections had occurred, mouse sera were tested periodically for seroconversion to mouse hepatitis virus andSendai virus by the ELISA test (Biocon Labs, Rockville, MD).

Three different recombinant IFNs were used. The recombinant hybrid human IFN-aA/D (rHuIFN-a),which is active in mouse cells and mice,'10' was kindly supplied by Drs. Michael Brunda and Peter Sorter(Hoffman-LaRoche). Recombinant mouse IFN-ß (rMuIFN-ß) was provided by courtesy of Dr. MasamiMoriyama (Toray Industries), and murine recombinant IFN-7 (rMuIFN-7) by Dr. Christine Czarniecki

'Department of Microbiology and Immunology, The Medical College of Pennsylvania, Philadelphia, PA 19129; and2The Wistar Institute, Philadelphia, PA 19004. 'Current address: Biology Department, Georgia State University, Atlanta,GA 30303.

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(Genentech). IFN titers were measured in a microplate cytopathic effect inhibition assay modified from themethod of Havell and Vilcek"" with encephalomyocarditis virus and mouse L-929 cells. All titres werecorrected against the NIH mouse reference standard preparation of IFN-a/ß (lot #86068, courtesy of LeeBiomolecular Research, San Diego, CA), even though strictly this may not be appropriate for nonglycosy-olated rMuIFN-ß or the human hybrid IFN-aA/D. The titers for rMuIFN--y were found to be the same whenadjusted against this standard or a reference mouse IFN-7 standard. Each IFN preparation was titrated oneach occasion it was used in mice, and from the results successive running mean titers were computed. Therunning mean titer at any time was used to calculate the dilution of that IFN to be used in the next animalstudy; from this dilution, and from the next running mean titer calculated incorporating the result from theparallel IFN assay, the amount of IFN used in that study was calculated. These amounts are shown in thetables.

IFN was diluted in Dulbecco's phosphate-buffered saline (DPBS) (GIBCO, Grand Island, NY) containing0.2% low endotoxin bovine serum albumin (BSA) (Sigma, St. Louis, MO) and injected intraperitoneally(¡•p.).

Virus stocks were prepared as previously described/6' The HSV-2 (strain MS) stock titered 107plaque-forming units (pfu)/ml on Vero cells; the Banzi flavivirus (strain SA M336), 2 x 106 pfu/ml on BHK15 cells; the SFV virus (strain L10), 6.8 x 107 pfu/ml on BHK 21 cells; and the Caraparu virus had an LD50of 102 3 in adult CD-I mice inoculated by the i.p. route.

For antiviral protection experiments there were generally 10 mice in each experimental group and 15 micein each placebo control group. A small LD50 dose-response assay was usually included in each experiment,and where applicable, a simultaneous plaque titration of the experimental virus dilution was also performed.Virus-infected mice were monitored daily for signs of illness [ruffled fur, hunched back, central nervous

system (CNS) disease symptoms] and for mortality. At the end of the test period (about 21 days for HSV-2and Banzi virus, and 14 days for Caraparu virus and SFV), the percent mortality and median survival time(MST) of both control and experimental groups was calculated.

Statistically significant differences (p < 0.05) in percent mortality were determined by the chi-squaretest. The MST was calculated, and the survival distribution data was analyzed using the Lee-Desu methodof group comparison which allows the most appropriate analysis of survival data with censored observations(i.e., mice still alive at the end of the observation period)/12' To simplify the tables, only the data andstatistical analysis for representative treatment groups are shown in comparison with respective controls. Therange of variability in the percent mortality and MST of the controls in the individual experiments are shownin the table footnotes.

Results obtained in the animal protection studies are summarized in Table 1.

(i) HSV-2 infections: There were no major differences in the antiviral effects of the three types of IFN withthe doses and regimens evaluated (Table 1 A). A single prophylactic dose (day -1 ), of any of the three IFNssignificantly reduced mortality from 90% in the control mice to 50%, and significantly increased the MST(p < 0.05); if treatment was continued through day +6 after infection, nearly all mice survived. Treatmentbegun 4 h after infection on day 0 and repeated daily through day +2 was also effective. There was still anantiviral effect when any of the IFNs was given only on days +1 and +2, or when the start of treatment was

delayed until day +2 and then continued until day +6.

(ii) Banzi virus infections: rMuIFN-7 given daily to infected mice from day 0 through +6, or day +1through +6, gave no protection (Table IB). However, when mice were treated on the same regimen withrHuIFN-a or rMuIFN-ß, significantly fewer died (p < 0.05); in contrast to the results with HSV-2infection, if the start of treatment was delayed until day +2, all protective effects were lost.

(iii) SFV infections: Regimens of pre- and post-infection treatment with any of the three IFN types were

highly effective (Table 1C). Repeated daily treatment from day 0 (the first dose at 4 h after infection) throughday +6, also gave protection. When given as a single dose at 4 h after infection, rHuIFN-aA/D was activebut not rMuIFN-7, and only the rHuIFN-aA/D preparation gave protection on a day +1 to day +6 schedule.

(iv) Caraparu virus infections: rHuIFN-aA/D and rMuIFN-ß gave no protection with the doses and

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schedules used. In contrast, rMuIFN-7 had some activity, but even so, its protective effects were less thanagainst any of the other three viruses tested (Table ID).

Our previous findings,'5,6' and those of others,<2~4,7"9' have shown that the viruses belonging to the fourdifferent virus families studied here, are all sensitive to the antiviral effects of one or more of the three typesof IFN. The present results extend these observations and are the first to compare the different IFN in a singlestudy.

Although IFNs have direct antiviral effects,' '3) they may also act indirectly in vivo through their effects onimmune mechanisms, for example on the activity of T lymphocytes, macrophages (M0), and natural killer(NK) cells, or by stimulating the production of other cytokines."3"15' To assess the relative importance ofthe various mechanisms in the studies reported here will require further work. Nevertheless, in thisconnection, it is of particular interest that only rMuIFN-7 was effective against Caraparu virus infections ofmice. This type of IFN is the most potent of the three as an activator of macrophages and NK cells,"5"18' andit appears also to differ from the others in the way it activates NK cells."9,201 However, other factors mayalso be important here, such as the major target organ involved in the different virus infections [in Caraparuvirus infections, the liver (unpublished observations); in infections with the other viruses studied, theCNS'21,22'] and how fast the infection develops (more rapidly with Caraparu virus than with the others).

The results of these mouse studies give some encouragement for the use of an IFN in human infectionswith these viruses, even though direct extrapolation from mouse to man is not possible and patients are

unlikely to receive IFN treatment before onset of clinical symptoms when the target organ(s) are likelyalready to be infected. Thus, perhaps an IFN will need to be used in conjunction with other agents in attemptsto treat these widespread and important virus infections of man.

ACKNOWLEDGMENTS

This work was supported by DMAD 17-86-6-6117. We wish to thank Drs. Michael Brunda and PeterSorter (Hoffman-LaRoche) for providing the rHuIFN-aA/D; Dr. Masami Moriyama (Toray Industries) forproviding the rMuIFN-ß; Dr. Christine Czarniecki (Genentech) for providing the rMuIFn-"y; Dr. DonnaMurasko for reviewing the manuscript and providing the IFN titers; and Dr. Edward Gracely for thestatistical analysis.

REFERENCES

1. DOWNS, W.G. (1982). Arboviruses, in: Viral Infections of Humans. S.A. Evans (ed.). New York: Plenum, pp.95-126.

2. CANÓNICO, P.G., KENDE, M., LUSCRI, B.J., and HUGGINS, J.W. (1984). ln-vivoactivity of antivirals againstexotic RNA viral infections. J. Antimicrob. Chemotherap. 14, Suppl. A, 27-41.

3. FINTER, N.B. (1973). Interferons and Inducers in vivo. I. Antiviral effects in experimental animals, in: Interferonsand Interferon Inducers. N.B. Finter, (ed.). Amsterdam: North Holland Publishing Company, pp. 295-362.

4. KENDE, M, LUPTON, H.W., RILL, W.L., GIBBS, P., LEVY, H.B., and CANÓNICO, P.G. (1987). Ranking ofprophylactic efficacy of poly(ICLC) against Rift-Valley fever virus infection in mice by incremental relative risk ofdeath. Antimicrob. Agents Chemother. 31, 1194-1198.

5. MORAHAN, P.S., LEAKE, E.R., TENNEY, DJ., and SIT, M.S. (1986). Comparative analysis of modulators ofnonspecific resistance against microbial infections, in: Immunologie Adjuvants and Modulators of NonspecificResistance to Microbial Infection. J. Majde, (ed.). New York: Alan R. Liss. pp. 313-324.

6. PINTO, A.J., MORAHAN, P.S., and BRINTON, M.A. (1988). Comparative study of various immunomodulatorsfor macrophage and natural killer cell activation and antiviral efficacy against exotic RNA viruses. Int. J.Immunopharmacol. 10, 197-209.

7. SARZOTTI, M., COPPENHAVER, D.H., SINGH, I,P., POAST, J., and BARON, S. (1989). The in vivo antiviraleffect of CL246,738 is mediated by the independent induction of IFN-a and IFN-ß. J. Interferon Res. 9, 265-274.

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8. STEWART II, W.E., SCOTT, W.D., and SULKIN, S.E. (1969). Relative sensitivities of viruses to different speciesof interferon. J. Virol. 4, 147-153.

9. TS'O, P.O.P., ALDERFER, J.L., LEVEY, J., MARSHALL, L.W., O'MALLEY, J., HOROSZEWICZ, J.S., andCARTER, W.A. (1976). An integrated and comparative study of the anti-viral effects and other biological propertiesof polyinosinic acid-polycytidylic acid and its mismatched analogues. Mol. Pharmacol. 12, 299-312.

10. KRAMER, M.J., DENNIN, R., KRAMER, C, JONES, G., CONNELL, E., ROLON, N., KALE, R., andTROWN, P.W. (1983). Cell and virus sensitivity studies with recombinant human alpha interferons. J. InterferonRes. 3,425-435.

11. HAVELL, E., and VILCEK, J. (1972). Production of high titered interferon in cultures of human diploid cells.Antimicrobial Agents Chemother. 2, 476-484.

12. LEE, E.,andDESU, M. (1972). A computer program for comparing k samples with wright-censored data. ComputerPrograms Biomed. 12, 315-321.

13. HARTSHORN, K.L., and HIRSCH, M.S. (1988). Interferons, in: The Antimicrobial Agents Annual 3. P. K.Peterson, and J. Verhoef (ed.). New York: Elsevier Science Publishers, pp. 371-383.

14. DAURAT, V., DORFMAN,P., and BASTIDE, M. (1988). Immunomodulatory activity of low doses of interferona, ß in mice. Biomed. Pharmacother. 42, 197-206.

15. MURRAY, H.W., SPITALNY, G.L., and NATHAN, CF. (1985). Activation of mouse peritoneal macrophages invitro and in vivo by interferon-7. J. Immunol. 134, 1619-1622.

16. MURRAY, H.W. (1988). Interferon-gamma, the activated macrophage, and host defense against microbialchallenge. Ann. Int. Med. 108, 595-608.

17. VARESIO,L.,BLASI,E.,THURMAN,G.B.,TALMADGE,J.E.,WILTROUT,R.H.,andHERBERMAN,R.B.(1984). Potent activation of mouse macrophages by recombinant interferon 7. Cancer Res. 44, 4465-4469.

18. WILSON, C.B., and WESTALL, J. (1985). Activation of neonatal and adult human macrophages by alpha, beta,and gamma interferons. Infect. Immun. 49, 351-356.

19. McGINNES, K., CHAPMAN, G., and PENNY, R. (1988). Effects of interferon on natural killer cells assessed byfluorescent probes and flow cytometry. J. Immun. Meth. 107, 129-136.

20. GUSTAFSSON, A., SUNDSTRÖM, S., and LUNDGREN, E. (1986). The augmentation of human natural killercell activity by interferon 7 is not associated with the induction of the interferon-a-inducible proteins. J. Immunol.137,167-171.

21. DOHERTY, P.C. (1973). Quantitative studies of the inflammatory process in fatal viral meningoencephalitis. Am.J. Pathol. 73, 607-622.

22. JACOBY, R.O., and BHATT, P.N. (1976). Genetic resistance to lethal flavivirus encephalitis. I. Infection ofcongenie mice with Banzi virus. J. Infect. Dis. 134, 158-165.

Address reprint requests to:Dr. Angelo J. Pinto

Department of Microbiology and ImmunologyThe Medical College of Pennsylvania

3300 Henry AvenuePhiladelphia, PA 19129

Received 10 April 1989/Accepted 16 November 1989

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