Acid-Poly-L-Lysine Complex on Encephalomyocarditis Virus

Transcription

1 ANTimICROsIAL AozWu AND CHEMoTHERAPY, Oct. 1976, p Copyright C 1976 Americn Society for Microbiology Vol. 10, No. 4 Printed in U.S.A. Effect of Tretment with Exogenous Interferon, Polyinosinic Acid-Polycytidylic Acid, or Polyinosinic Acid-Polycytidylic Acid-Poly-L-Lysine Complex on Encephlomyocrditis Virus Infections in Mice' GREGG A. OLSEN,* EARL R. KERN, LOWELL A. GLASGOW, AND JAMES C. OVERALL, JR. Deprtments of Peditrics* nd Microbiology, University of Uth, College of Medicine, Slt Lke City, Uth Received for publiction 12 April 1976 The effect of tretment with exogenous interferon ws compred with two interferon inducers, polyinosinic cid-polycytidylic cid [poly(i:c)] nd [poly(i:c)ipoly-l-lysine complex (P-L-L complex), in two model encephlomyocrditis virus infections of mice. Although both inducers stimulted the production of interferon, the pek serum levels induced by P-L-L complex were five- to eightfold greter thn those induced with poly(i:c). When encephlomyocrditis virus ws inoculted by either the intrperitonel or the intrnsl route, interferon nd both of the inducers protected mice ginst mortlity nd prolonged the men dy of deth when the compounds were given prior to or immeditely fter virl chllenge. In generl, tretment with interferon ws not s successful s tretment with poly(i:c) or P-L-L complex. In these infections, P-L-L complex ppered to be the most effective gent in tht successful tretment resulted when drug therpy ws initited s lte s 48 h fter virus inocultion. An exmintion of the effect of tretment on the pthogenesis of the infection indicted tht protection ws ssocited with the prevention of viremi nd subsequent seeding of trget orgns, prticulrly the centrl nervous system. Enteroviruses re frequent cuse of humn infections nd result in wide spectrum of disese, including nonspecific febrile illness, respirtory infections, septic meningitis, encephlitis, myocrditis, or pericrditis (16, 21, 23). The infection of mice with encephlomyocrditis (EMC) virus ppers to simulte some humn enterovirus infections in tht EMC virus is murine picornvirus, trget orgns re seeded by viremi, nd deth results primrily from the involvement of the centrl nervous system (25). This murine infection, therefore, provides model for the evlution of ntivirl chemotherpeutic gents tht my hve potentil for use in the tretment of humn disese. Interferon is thought to be n importnt component of host resistnce to virl infections. Results from number of lbortories hve suggested tht both interferon (9, 10, 18) nd interferon inducers (2-4, 11, 14, 15) my be used successfully in the prevention or tretment of certin virl infections in experimentl nimls nd in humns. Although exogenous interferon hs been evluted in severl clinicl ' Publiction no. 26 from the Coopertive Antivirl Testing Group Ntionl Institute of Allergy nd Infectious Disese. trils in humns (6, 17, 19, 27), controlled studies in nimls hve been reltively limited. Becuse high-titered preprtions of mouse interferon hve recently become vilble, we compred the efficcy of interferon nd interferon inducers ginst experimentl EMC virus infections in this species. The most common interferon inducer used in murine virl infections, polyinosinic cid-polycytidic cid [poly- (I:C)], stimultes reltively low levels of interferon in humns, presumbly becuse of the rpid hydrolysis tht occurs when poly(i:c) is mixed with primte serum (24). A new interferon inducer, complex of poly(i:c) with poly- Irlysine (P-L-L complex), is 5 to 10 times s resistnt to this hydrolysis s poly(i:c) (22) nd induces significnt levels of interferon in the serum of monkeys under conditions when poly(i:c) does not. The purpose of this study ws to compre the therpeutic efficcy of exogenous murine interferon with the two interferon inducers, poly(i:c) nd P-L-L complex, in the tretment of two experimentl EMC virus infections in mice. The effect of tretment ws evluted by determining the ltertion of mortlity, men dy of deth, nd pthogenesis of the infection. 668

2 VOL. 10, 1976 MATERIALS AND METHODS Animls nd experimentl infections. Sevenweek-old mice were obtined from Simonsen Lbortories, Gilroy, Clif. Animls were inoculted by either the intrperitonel (i.p.) or intrnsl route. Mice infected by the i.p. route were given pproximtely 50 plque-forming units (PFU) (3 men lethl doses [LD.,]) of EMC virus, which resulted in n 80 to 90% mortlity. In the intrnsl infection, ether-nesthetized mice were llowed to inhle 0.05 ml of virus suspension contining pproximtely 2.7 x 104 PFU (3 LD5,), which resulted in 75 to 85% mortlity. All nimls were observed for 21 dys fter virus inocultion. Virus. The strin of EMC virus used in these studies ws lrge plque vrint originlly obtined from K. K. Tkemoto (Ntionl Institutes of Helth, Bethesd, Md.). Virus pools were prepred by hrvesting brins from infected suckling mice nd titered 107 PFU/ml on L-cell fibroblst cultures. Vesiculr stomtitis virus (VSV), Indin strin, ws obtined from the Americn Type Culture Collection (Rockville, Md.), nd stock pools were prepred in primry chicken embryo cells s described previously (25). Medi, cell cultures, virus ssys, nd interferon ssys. The medi utilized, the mintennce of mouse L-cell (L-929) fibroblst cultures, nd the ssy for EMC virus hve been described in previous publiction (25). Interferon ws ssyed by using plque reduction method with VSV s the chllenge virus s described previously (20). A reference mouse serum interferon preprtion ws included in ech ssy. Interferon nd interferon inducers. All compounds being evluted were provided by the Antivirl Substnces Testing Progrm, Ntionl Institute of Allergy nd Infectious Diseses, Bethesd, Md. Exogenous interferon ws prepred in mouse L- 929 cells either by Kurt Pucker t the Medicl College of Pennsylvni, Phildelphi, P., or by Bionetics Lbortories, Kensington, Md. The interferon prepred by Dr. Pucker ws received s lyophilized powder nd stored t -70 C (per his instructions) until diluted for use to n pproprite concentrtion with phosphte-buffered sline (PBS). Bionetics interferon ws received s frozen liquid t concentrtion of 106 U/ml nd stored t -30 C until use (per mnufcturer's recommendtion). Poly(I:C), prepred by P-L Biochemicls, Inc., Milwukee, Wis., hd concentrtion of 1 mg/ml nd ws stored t -20 C until use. The P-L-L complex ws prepred by H. B. Levy t the Ntionl Institute of Allergy nd Infectious Diseses. Poly(I:C) (2 mg/ml) ws complexed with poly-l-lysine (1.5 mg/ ml) in 0.5% crboxymethyl cellulose in sline (22). This solution ws stored t 4 C nd diluted with n equl volume of PBS t the time of use. All drugs were dministered in 0.1-ml volume by the i.p. route. Virus control nimls received 0.1 ml of PBS dministered i.p. on the sme tretment schedules. Sttisticl evlution. The differences in mortlity between nimls treted with drug nd control nimls receiving PBS were evluted by chisqure nlysis. To compre the men dy of deth TREATMENT OF EMC VIRUS INFECTIONS IN MICE 669 of drug-treted nd PBS-treted control nimls, the dt were evluted by the use of the Student t test. A P vlue of c0.05 ws considered to be significnt. RESULTS Interferon induction. The levels of interferon in the serum of mice fter the injection of 100 ug of either poly(i:c) or P-L-L complex re shown in Tble 1. Pek titers occurred 9 h fter injection with both compounds. The response induced by P-L-L complex ws pproximtely five times higher nd lsted 24 h longer thn tht induced by poly(i:c). Interferon induction fter repeted injections. The levels of interferon in the serum of mice fter multiple injections of 100 ug of either poly(i:c) or P-L-L complex dministered ccording to the tretment schedules utilized in this series ofexperiments re shown in Tble 2. Poly(IC) induced lower, but still substntil, level of serum interferon fter the second injection, but with subsequent injections hyporectivity of the interferon response occurred. In contrst, lthough P-L-L complex induced greter serum interferon response thn did poly(i:c) fter the first injection, the second injection filed to induce more thn 900 to 1,200 U of interferon per ml. The dt in Tble 2 demonstrte tht the kinetics of the interferon response re not chnged with multiple doses of either inducer. Effect of tretment on mortlity of mice inoculted i.p. with EMC virus. The effect of tretment with exogenous interferon, poly(i:c), or P-L-L complex on the mortlity nd men dy of deth of mice infected i.p. with EMC virus is shown in Tble 3. The results in the treted groups in seprte experiments were compred with the results in control groups pooled from ll experiments. The virus-infected control nimls, treted with PBS i.p. twice TABLz 1. Titers of interferon in the serum of dult mice inoculted i.p. with poly(i:c) or P-L-L complex Titee of interferon Time (h) Poly(I:C) P-L-L complex (100,ug) (100 /Ag) 3 3,850 1, ,750 23, ,275 25, ,400 18, , < < Interferon titers re expressed s the reciprocl of the highest dilution tht inhibited 50% of the VSV chllenge.

3 670 OLSEN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Titers of interferon in the serum of dult mice repetedly inoculted i.p. with poly(i:c) or P-L-L complex Titer" of interferon on dy fter Inducer Time initition of tretment: (h) Poly(I:C) given on dys 0, 1, 3, 6 6,465 4,055 1,585 1,620 3,735 4, nd , ,245 NS" 2, < P-L-L complex given on dys 0, 6 30, ,195 2, 4, nd , NS , , < Interferon titers re expressed s the reciprocl of the highest dilution tht inhibited 50% of the VSV chllenge. b NS, No smple. TABLE 3. Effect of tretment with exogenous interferon, poly(i:c), or P-L-L complex on the mortlity nd men dy of deth in dult mice infected i.p. with EMC virus Mortlity Tretment Men dy of deth P vlue No. % P vlue None 33/ Exogenous interferon Expt 1b -3 h 1/15 7 < h 1/14 7 s h 9/15 60 NSC 5.6 NS Drug controlb 1/ Expt 2d -1 h 0/15 0 s h 2/15 13 s h 11/15 73 NS 5.5 NS Drug controld 0/15 0 Poly(I:C)e -6 h 1/25 4 s h 1/24 4 s h 10/25 40 < NS Drug controle 0/25 0 P-L-L- complexf -24 h 1/25 4 s h 8/ ' h 10/25 40 ' NS Drug controy 0/25 0 Tretment ws initited t the indicted times prior to or fter virl chllenge. b 20,000 U/mouse ws given twice dily for 7 dys (Pucker). c NS, Not significnt. d 40,000 U/mouse ws given twice dily for 7 dys (Pucker). e 100,Ag/mouse ws given on the bsis of once dily for 2 consecutive dys nd then skipping dy, for 7 dys. f 100,ug/mouse ws given once every other dy for 7 dys. dily for 7 dys, exhibited n 83% finl mortlity, with men dy of deth of 5.7 dys. Infection ws chrcterized by hunching nd circling by dy 3, hind limb prlysis by dy 4, nd deth by dy 5 to 7 fter virl inocultion. In experiment 1 tretment consisted of 20,000 U of exogenous interferon dministered twice dily for 7 dys. Tretment initited 3 h prior to

4 VOL. 10, 1976 or 1 h fter virl inocultion reduced the finl mortlity to only 7% (P 0.001). Therpeutic - efficcy ws lost when tretment ws initited 24 h fter virus inocultion. In experiment 2 the dose ws incresed to 40,000 U of interferon given i.p. twice dily for 7 dys. The results of therpy with this incresed dose of interferon were similr to those of experiment 1 in tht tretment ws highly effective when initited prior to or 1 h fter, but not 24 h fter, infection. Although repeted injections of poly(i:c) were shown to result in progressive hyporectivity of the interferon response, previous dt from our lbortory indicted tht tretment of mice infected with EMC virus, utilizing multiple doses of poly(i:c), ws more effective thn single dose (26). Bsed on these dt, therpy consisted ofsix doses of 100 ig of poly(i:c) dministered on dys 0, 1, 3, 4, 6, nd 7 ofthe infection. Becuse the serum interferon response to P-L-L TREATMENT OF EMC VIRUS INFECTIONS IN MICE 671 complex reched higher levels nd lsted longer, 100,ug ws dministered i.p. every other dy for 7 dys ( totl of four doses). Tretment with either poly(i:c) or P-L-L complex resulted in significnt reduction in mortlity, even when the initition of therpy ws delyed until 24 h fter virl chllenge. Effect of tretment on mortlity of mice inoculted intrnslly with EMC virus. To pproximte more nturl route of infection, mice were inoculted intrnslly with EMC virus. This experimentl infection ws lso chrcterized by hunching nd hind limb prlysis by dy 4, followed by deth on dys 5 to 7 of infection. The effect of exogenous interferon, poly(i:c), or P-L-L complex tretment on the mortlity nd men dy of deth of mice infected intrnslly with EMC virus is shown in Tble 4. The sme therpeutic regimens s described bove were used for ech compound nd TABLE 4. Effect of tretment with exogenous interferon, poly(i:c), or P-L-L complex on the mortlity nd men dy of deth in dult mice infected intrnslly with EMC virus Mortlity Tretment" Men dy of deth P vlue No. % P vlue None 32/ Exogenous interferon Expt lb -3 h 1/ h 1/ h 2/ Drug contropb 0/10 0 Expt 2c +1 h 6/ h 6/10 60 NSd h 15/20 75 NS 5.5 NS Drug controle 0/20 0 Poly(I:C)e -6 h 0/ h 1/20 5 < h 6/ NS +48 h 7/10 70 NS 5.4 NS Drug controle 0/20 0 P-L-L complex' -24 h 0/10 0 < h 6/29 21 < s h 3/20 15 < h 9/20 45 s s0.01 Drug controlf 0/20 0 Tretment ws initited t the indicted times prior to or fter virl chllenge. b 40,000 U/mouse ws given twice dily for 7 dys (Pucker). c 100,000 U/mouse ws given twice dily for 7 dys (Bionetics; see text). d NS, Not significnt. e100 lug/mouse ws given on bsis of once dily on 2 consecutive dys nd then skipping dy, for 7 dys. ' 100,ug/mouse ws given once every other dy for 7 dys.

5 672 OLSEN ET AL. the results of seprte experiments re presented. The control group represents dt pooled from ll of the experiments. The virusinfected control nimls treted i.p. with PBS twice dily for 7 dys exhibited n 80% finl mortlity (men dy of deth, 5.3 dys). In experiment 1 tretment with 40,000 U of interferon (Pucker) twice dily for 7 dys beginning 3 h prior to infection resulted in finl mortlity of 10% (P < 0.001) nd men dy of deth of 6.0 dys. In contrst to the results. of the tretment of the infection initited by the i.p. route, therpy beginning either 1 or 24 h fter virl inocultion were both highly effective in reducing mortlity. To determine whether higher doses of interferon would increse efficcy, dose of 100,000 U twice dily of interferon (Bionetics) ws used in the second experiment. Initition of therpy t 1 h fter infection resulted in similr protection, wheres therpy strted t either 24 or 48 h fter infection did not significntly reduce the finl mortlity. Although both interferon preprtions were effective when tretment ws initited t 1 h fter infection, the Bionetics preprtion ws less effective with tretment beginning t 24 h. Even though the Bionetic interferon preprtion titered pproximtely 106 U/ml t the beginning of the experiments, ssys performed 15 weeks lter, or 6 weeks fter the completion of these studies, indicted tht the titers hd decresed 10- to 50-fold. Repet titrtions with the Pucker interferon reveled no deteriortion in titer. Therpy with poly(i:c) initited 6 h prior to infection completely protected ll nimls, nd tretment initited 1 or 24 h fter infection significntly reduced the finl mortlity. Therpeutic efficcy ws lost when tretment ws begun 48 h fter infection. Tretment with P-L- L complex begun either prior to or t 1 or 24 h fter virl chllenge fforded similr degree ofprotection s did poly(i:c). In ddition, tretment with P-L-L complex initited s lte s 48 h fter infection significntly protected mice, s well s incresed the men dy of deth. Effect of tretment on EMC virus pthogenesis. In n ttempt to elucidte the mechnism of protection by interferon in EMC virusinfected mice, the effect of drug tretment on the pthogenesis of the infection ws defined. In seprte experiments, nimls were infected intrnslly nd treted with exogenous interferon or P-L-L complex beginning either t 1 or 48 h fter infection. Three individul mice from ech group were scrificed dily nd bled by crdic puncture, nd the blood ws homogenized undiluted. The lungs, liver, spleen, hert, nd brin were removed nd homogenized s 10% suspensions in miniml essentil ANTIMICROB. AGENTS CHEMOTHER. medium with 10% fetl bovine serum. Tissue homogentes from the individul nimls were centrifuged, superntnts were collected, nd ll smples were stored t - 70 C until ssyed for virus. The effect of 100,000 U of exogenous interferon (Bionetics) given twice dily for 5 dys on the pthogenesis ofemc virus infection is summrized in Tble 5. It should be noted tht the deth rte in control group of mice followed for mortlity ws 90%, indicting tht not ll nimls in ech group becme infected. No virus could be detected on dy 1 ofthe infection in ny of the control nimls treted with PBS. By dy 2 ofthe infection, 104 to 106 PFU ofemc virus per g were isolted from the lungs nd hert, 103 to 104 PFU/g were isolted from the liver nd spleen, nd 102 to 104 PFU/ml were isolted from the blood in two nimls. No virus ws isolted from the third niml. On dy 3, virus ws detected t levels of 104 to 106 PFU/g in multiple tissues in one niml, but only in lung tissue in the second niml. Virus ws not recovered from the third niml. On dy 4, ll nimls were infected nd titers of virus from 104 to 107 PFU/g were recovered from multiple tissues. In nimls treted with interferon beginning t 1 h fter infection, mrked reduction in the recovery of virus from tissues ws observed. Only 4 ofthe 12 smpled nimls demonstrted evidence of infection with EMC virus. The deth rte in compnion group of nimls treted with the sme regimen nd followed for mortlity ws 33%. When interferon therpy ws begun t 48 h fter virl inocultion, time when the viremi hd occurred nd high titers of virus were lredy present in the orgns of infected nimls, the pthogenesis of infection ws similr to tht of PBS-treted control nimls. The mortlity ws 80% in compnion group of nimls treted with the sme regimen. These dt correlte with the protection ginst mortlity observed when the tretment ws initited t 1 h fter infection nd with the bsence of protection when the tretment ws initited t 48 h fter infection. The effect of 100 ug of P-L-L complex given once every other dy for 5 dys on the pthogenesis of intrnsl EMC virus infection of mice is summrized in Tble 6. In this experiment ll three virus control nimls demonstrted evidence of virl repliction in lung tissue on dy 1, nd six of the nine control nimls hrvested on dys 2, 3, nd 4 were infected with virus. The deth rte in control group of nimls ws 80%o, gin indicting tht not ll nimls in ech group were infected. In contrst, in the group of nimls in

6 VOL. 10, 1976 TREATMENT OF EMC VIRUS INFECTIONS IN MICE 673 TABLE 5. Effect oftretment with exogenous interferon on the pthogenesis ofintrnsl EMC virus infection Titer of EMC virus on dy: Tissue exmined Virus control Blood <1, <1, <1 <1, 2.8, 4.4 <1, <1, 4.2 <1, 6.0, <1 Brin <2, <2, <2 <2, <2, <2 <2, <2, , 7.6, 6.0 Hert <2, <2, <2 <2, 5.0, NSb <2, <2, , 6.0, 6.0 Lung <2, <2, <2 <2, 6.3, , <2, , 6.0, 5.8 Liver <2, <2, <2 <2, 3.5, 3.9 <2, <2, 3.5 <2, 5.4, 5.0 Spleen <2, <2, <2 <2, 3.3, 4.4 <2, <2, , 5.6, 4.1 Tretment initited t 1 he Blood <1, <1, 3.3 <1, <1, 6.0 <1, <1, <1 <1, <1, <1 Brin <2, <2, <2 <2, <2, <2 <2, <2, <2 <2, <2, <2 Hert <2, <2, <2 <2, <2, 6.0 <2, <2, <2 3.4, 3.3, <2 Lung <2, <2, 5.4 <2, <2, 6.0 <2, <2, <2 3.8, 3.8, <2 Liver <2, <2, <2 <2, <2, 5.0 <2, <2, <2 4.0, 3.2, <2 Spleen <2, <2, <2 <2, <2, 4.5 <2, <2, <2 4.3, 3.4, <2 Tretment initited t 48 he Blood <1, 2.9, <1 <1, <1, , 3.9, <1 3.7, 3.6, <1 Brin <2, <2, <2 <2, <2, <2 NS, 5.9, <2 5.6, 5.2, <2 Hert 4.2, 5.7, <2 4.4, <2, , 4.2, <2 6.3, 5.7, 3.5 Lung 6.2, 6.0, , <2, , 3.4, <2 5.7, 3.4, 4.1 Liver <2, 3.2, <2 <2, <2, 3.4 <2, 2.9, <2 3.4, 4.9, <2 Spleen 2.4, 3.0, <2 <2, <2, 3.3 <2, 4.6, <2 4.6, 4.3, <2 Titers of EMC virus in orgns of nimls from ech group. Virl titers re expressed s log1o PFU per grm of tissue or log1o PFU per milliliter of blood. Ech column indictes virl titers in orgns of one niml. b NS, No smple. c 100,000 U/mouse ws given twice dily for 5 dys (Bionetics; see text). TABLE 6. Effect of tretment with P-L-L complex on the pthogenesis of intrnsl EMC virus infection Titer of EMC virus on dy: Tissue exmined Virus control Blood <1, <1, <1 1.4, 2.2, <1 1.7, <1, <1 <1, <1, 5.5 Brin <2, <2, <2 4.4, 3.8, <2 1.7, <2, <2 <2, <2, 8.0 Hert <2, <2, <2 4.1, 4.0, <2 4.5, <2, <2 5.7, 2.6, <2 Lung 3.9, 4.1, , 3.8, <2 4.1, <2, <2 <2, <2, 5.9 Liver <2, <2, <2 <2, <2, <2 3.2, <2, <2 <2, <2, 6.0 Spleen <2, <2, <2 3.6, 2.9, <2 <2, <2, <2 <2, 3.8, 6.0 Tretment initited t 1 hb Blood <1, <1, <1 <1, <1, <1 <1, <1, <1 <1, <1, <1 Brin <2, <2, <2 <2, <2, <2 <2, <2, <2 <2, <2, <2 Hert <2, <2, <2 <2, <2, <2 <2, <2, <2 <2, <2, <2 Lung <2, 2.7, , <2, <2 <2, <2, <2 <2, <2, <2 Liver <2, <2, <2 <2, <2, <2 <2, <2, <2 <2, <2, <2 Spleen <2, <2, <2 <2, <2, <2 <2, <2, <2 <2, <2, <2 Tretment initited t 48 hb Blood <1, <1, <1 1.4, 4.2, , <1, <1 <1, <1, 2.9 Brin <2, <2, <2 <2, <2, <2 <2, <2, <2 <2, <2, 7.5 Hert <2, <2, <2 3.3, 4.1, , <2, <2 <2, 3.8, 5.6 Lung <2, 5.2, , 4.1, , 3.5, <2 <2, 4.9, 5.1 Liver <2, <2, <2 3.0, 3.9, <2 <2, <2, <2 <2, 2.7, 5.1 Spleen <2, <2, <2 4.1, 4.1, <2 <2, <2, <2 <2, 5.0, 4.1 Titers of EMC virus in orgns of nimls from ech group. Virl titers re expressed s log1o PFU per grm of tissue or log1o PFU per milliliter of blood. Ech column indictes virl titers in orgns of one niml. b 100,ug/mouse ws given once every other dy for 5 dys.

7 .674 OLSEN ET AL. which therpy with P-L-L complex ws begun t 1 h, virl repliction ws observed in only 3 of 12 nimls. Only lung tissue ppered to be involved erly in the course of the infection. The deth rte in compnion group ofnimls treted with the sme regimen nd followed for mortlity ws 30%. In the group of mice tht received tretment beginning t 48 h, three of the six nimls exmined on dys 3 nd 4 demonstrted either no virus in tissues or virus tht ws confined to lung tissue. In the remining treted nimls the virus titers in tissues were similr to those observed in the virus control group. Mortlity ws 50% in compnion group of nimls treted with the sme regimen. These dt correlte with protection ginst mortlity observed when the tretment ws initited t 1 h fter infection nd with the fct tht pproximtely hlf of the mice were protected when P-L-L complex ws initited t 48 h fter virus infection. DISCUSSION It hs frequently been reported tht the dministrtion of exogenous interferon or interferon inducers cn reduce the mortlity of nimls inoculted with viruses tht infect the centrl nervous system (7, 9, 12, 13, 16, 25, 28). In generl, these studies hve lso demonstrted tht these compounds re most effective when dministered prior to or immeditely fter inocultion of virus. With the recent vilbility of high-titered preprtions of murine interferon nd the development of P-L-L complex, n interferon inducer tht stimultes higher serum levels of circulting interferon in mice nd primtes thn poly(i:c) (22), we compred the ntivirl effect of these compounds in experimentl EMC virus infections of mice to determine whether the chievement of higher levels of interferon would result in incresed therpeutic efficcy. In two model EMC virus infections, ll three compounds (interferon, poly(i:c), nd P-L-L complex) were effective in preventing mortlity iftretment ws begun t 1 h fter virl chllenge. Of the compounds evluted in this study, P-L-L complex ws the most effective in tht tretment could be delyed until 48 h fter intrnsl inocultion, wheres efficcy ws lost fter 24 h with either exogenous interferon or poly(i:c). This incresed effectiveness is likely due to the fct tht P-L-L complex induces higher nd longer lsting levels of serum interferon. Previous studies (3, 5, 12, 13, 28) oftretment with interferon or interferon inducers in EMC virus infections hve not demonstrted s gret reduction in mortlity s noted in the current study. ANTIMICROB. AGENTS CHEMOTHER. This my reflect our use of lower virl inoculum (3 LD.,,), which resulted in only 80 to 90%o mortlity, nd the employment of lrger doses of interferon or interferon inducers. Incresing the dose of exogenous interferon from 40,000 to' 80,000 U dy did not significntly improve protection ginst i.p. chllenge with EMC virus. There re severl possible explntions for this observtion. First, doubling the dose of interferon my not provide significnt increse in interferon levels within trget orgns nd considerbly lrger doses my be required in order to further increse efficcy. Second, the possibility exists tht there is plteu beyond which incresed concentrtions of interferon do not give incresed protection. Further studies re needed to determine which, if either, ofthese two possibilities re correct. In these experiments we utilized two preprtions of exogenous interferon which were produced by the sme technique, but in two different lbortories. When tretment ws initited t 1 h fter intrnsl infection with EMC virus, there ws no significnt difference in the finl mortlity of mice tht received 80,000 U dily of the Pucker preprtion of interferon s compred with those tht were given 200,000 U dily of Bionetics interferon. If therpy ws initited t 24 h fter infection, however, dily tretment with 80,000 U of the Pucker preprtion resulted in significnt protection, wheres tretment with 200,000 U dily of Bionetics interferon did not. The reduced effect of the Bionetics preprtion my hve been due to the decrese in titer tht we observed fter the completion of these experiments. Although we dhered to the storge conditions recommended for both preprtions of interferon, it is possible tht the frozen liquid interferon provided by Bionetics ws less stble thn the lyophilized preprtion from Dr. Puker. In n effort to elucidte the mechnisms of protection, the effect of therpy on the pthogenesis of the infection in individul nimls ws defined. In our previous studies of systemic EMC virus infection in mice, it ws demonstrted tht protection due to tretment with poly(i:c) or tilorone hydrochloride correlted with the complete suppression of viremi nd prevention of seeding of trget orgns (25, 26). In the present study utilizing n intrnsl inocultion of EMC virus, repliction occurred in the lungs by 24 to 48 h, nd viremi nd seeding of the brin, hert, liver, nd spleen were detected by 48 h fter the initition of infection. The erly presence of circulting interferon (1 h tretment), whether dministered

8 VOL. 10, 1976 exogenously or induced by P-L-L complex, inhibited virl repliction in the lungs nd prevented the development of the subsequent viremi nd seeding of trget orgns. This observtion would gree with the evidence tht protection due to circulting interferon is result of the inhibition of virl repliction t the initil site ofinfection (1, 7, 25, 26) nd suggests tht if sufficiently high levels of interferon re chieved virus repliction in trget orgns cn be completely inhibited. If therpy ws not initited until 48 h fter infection, tretment with exogenous interferon ws ineffective in preventing mortlity. The greter efficcy ofthe P- L-L complex, which induced higher initil levels of interferon in the serum of mice, ws evidenced by the cpcity of this inducer to suppress repliction of virus in the trget orgns of some nimls in which tretment ws initited s lte s 48 h. This observtion correltes with the fct tht bout 50% of the nimls in this group were protected. Our dt concerning the pthogenesis of this infection suggest tht the seeding of trget orgns occurs t pproximtely 48 h nd, thus, it ppers tht the levels of circulting interferon initilly induced by P-L-L complex were sufflcient to prevent virl repliction in the trget orgns of some nimls. It ppers, however, tht in spite of the high initil levels of interferon induced by P-L-L complex, once the virus infection ws estblished in the centrl nervous system therpy ws ineffective. This interprettion further supports the conclusions of De- Clercq nd Merign (5) nd Finter (8), who reported tht once the centrl nervous system of mice hd been seeded with VSV or Semliki forest virus, therpy with interferon inducers ws ineffective in preventing mortlity. The results of this study suggest tht the cpcity of interferon or interferon inducers to modify virl infection my be enhnced by producing higher levels of interferon in the infected host. On the other hnd, once virl repliction hs become estblished in criticl trget orgns, neither interferon inducers nor exogenous interferon ppered to exert beneficil ntivirl effects. The dt do suggest, however, tht for either exogenous interferon or interferon inducers to be optimlly effective, there is need to further increse the cpcity to dminister, or induce, higher nd more sustined levels of interferon. TREATMENT OF EMC VIRUS INFECTIONS IN MICE 675 ACKNOWLEDGMENTS This work ws supported by Public Helth Service contrct NOI-AI from the Antivirl Substnces Progrm of the Ntionl Institutes of Allergy nd Infectious Disese nd by Public Helth Service grnt AI from the Ntionl Institute of Allergy nd Infectious Disese. Jmes C. Overll, Jr., is n investigtor of the Howrd Hughes Medicl Institute. LITERATURE CITED 1. Bron, S., C. E. Buckler, R. M. Friedmn, nd R. V. McCloskey Role of interferon during viremi. II. Protective ction of circulting interferon. J. Immunol. 96: Ctlno, L. W., Jr., nd S. Bron Protection ginst herpes virus nd tencephlomyocrditis virus encephlitis with double-strnded RNA inducer of interferon. Proc. Soc. Exp. Biol. Med. 133: Ctlno, L. W., Jr., W. T. London, J. M. Rice, nd J. C. Sever Prophylctic nd therpeutic use of poly (I) * poly (C) (poly-d-lysine) ginst herpesvirus encephlitis in mice. Proc. Soc. Exp. Biol. Med. 140: De Clercq, E., nd P. De Somer Comprtive study of the efficcy of different forms of interferon therpy in the tretment ofmice chllenged intrnslly with Vesiculr Stomtitis Virus. Proc. Soc. Exp. Biol. Med. 138: De Clercq, E., nd T. C. Merign Locl nd systemic protection by synthetic polynionic interferon inducers in mice ginst intrnsl Vesiculr Stomtitus Virus. J. Gen. Virol. 5: Emodi, G., R. O'Reilly, A. Muller, L. K. Eberson, U. Binswnger, nd M. Just The effect of humn exogenous leukocyte interferon in cytomeglovirus infection. J. Infect. Dis.133(Suppl):A199-A Finter, N Protection of mice by interferon ginst systemic virus infections. Br. Med. J. 2: Finter, N. B Interferon s n ntivirl gent in vivo: quntittive nd temporl spects of the protection of mice ginst Semliki Forest virus. Br. J. Exp. Pthol. 47: Finter, N. B Interferon in nimls nd mn, p In. N. B. Finter (ed.), Interferons. North Hollnd Publishing Co. Amsterdm. 10. Finter, N. B Exogenous interferon in nimls nd its clinicl implictions. Arch. Intern. Med. 126: Gerone, P. J., D. A. Hill, L. H. Appell, nd S. Bron Inhibition of respirtory virus infections of mice with erosols of synthetic double-strnded ribonucleic cid. Infect. Immun. 3: Gresser, I., C. Bourli, M. T. Thoms, nd E. Flcoff Effect of repeted inocultion of interferon preprtions on infection of mice with encephlomyocrditis virus. Proc. Soc. Exp. Biol. Med. 127: Gresser, I., D. Fontine-Brouty-Boye, C. Bourli, nd M. T. Thoms A comprison of the efficcy of endogenous, exogenous, nd combined endogenousexogenous interferon in the tretment of mice infected with encephlomyocrditis virus. Proc. Soc. Exp. Biol. Med. 130: Hillemn, M. R Double strnded RNA's (poly I:C) in the prevention of virl infections. Arch. Intern. Med. 126: Hillemn, M. R Prospects for the use of doublestrnded ribonucleic cid (poly I:C) inducers in mn. J. Infect. Dis. 121: Horstmnn, D. M Enterovirus infections: etiologic, epidemiologic, nd clinicl spects. Clif. Med. 103: Jones, B. R., D. J. Coster, M. G. Flcon, nd K. Cntell Clinicl trils of topicl interferon therpy of ulcertive virl kertitis. J. Infect. Dis. 133(Suppl.):A169-A Jones, B. R., J. E. K. Glbrith, nd M. K. Al-Hussine Vccinil kertitis treted with inter-

9 676 OLSEN ET AL. feron. Lncet 1: Kufmn, H. E., R. F. Meyer, P. R. Libson, S. R. Wltmn, A. B. Nesburn, nd J. J. Shuster Humn leukocyte interferon for the prevention of recurrences of herpetic kertitis. J. Infect. Dis. 133(Suppl.):A165-A Kern, E. R., J. C. Overll, Jr., nd L. A. Glsgow Herpesvirus hominis infection in newborn mice: tretment with interferon inducer polyinosinic-polycytidylic cid. Antimicrob. Agents Chemother. 7: Kibrick, S Current sttus of Coxsckie nd ECHO viruses in humn disese. Prog. Med. Virol. 6: Levy, H. B., G. Buer, S. Bron, C. E. Buckler, C. J. Gibbs, M. J. Iodrol, W. T. London, nd J. Rice A modified polyribonosinic-polyribocytidylic cid complex tht induces interferon in primtes. J. Infect. Dis. 132: McLen, D. M Coxsckieviruses nd echoviruses. Am. J. Med. Sci. 251: ANTIMICROB. AGENTS CHEMOTHER. 24. Nordlund, J. J., S. M. Wolff, nd H. B. Levy Inhibition of biologic ctivity of poly I:poly C by humn plsm. Proc. Soc. Exp. Biol. Med. 133: Stringfellow, D. A., J. C. Overll, Jr., nd L. A. Glsgow Interferon inducers in therpy of infection with encephlomyocrditis virus in mice. I. Effect of single doses of polyinosinic-polycytidylic cid nd tilorone hydrochloride on virl pthogenesis. J. Infect. Dis. 130: Stringfellow, D. A., J. C. Overll, Jr., nd L. A. Glsgow Interferon inducers in therpy of infection with encephlomyocrditis virus in mice. I. Effect of multiple doses of polyriboinosinic-polyribocytidylic cid on virl pthogenesis. J. Infect. Dis. 130: Sundmcher, R., D. Neumnn-Hefelin, K. F. Mnthey, nd A. Muller Interferon in tretment of dendritic kertitis in humns: preliminry report. J. Infect. Dis. 133(Suppl.):A160-A Worthington, M., nd S. Bron Lte therpy with n interferon stimultor in n rbovirus encephlitis in mice. Proc. Soc. Exp. Biol. Med. 136: Downloded from on November 21, 2018 by guest