Inhibition of Respiratory Virus Infections of Mice with Aerosols of Synthetic Double-Stranded Ribonucleic Acid

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1 INFECriON AND IMMUNITY, Feb. 1971, p Copyright 1971 Americn Society for Microbiology Vol. 3, No. 2 Printed in U.S.A. Inhibition of Respirtory Virus Infections of Mice with Aerosols of Synthetic Double-Strnded Ribonucleic Acid PETER J. GERONE, DAVID A. HILL,1 LOREN H. APPELL,2 AND SAMUEL BARON Biologicl Scienices Lbortories, Fort Detrick, Frederick, Mrylnid 21701, nid Ntionl Intstitute of Allergy nd Inifectious Diseses, Bethlesd, Mrylnd Received for publiction 11 September 1970 Aerosols of double-strnded complexes of polyinosinic nd polycytidylic cids (poly I:C) were useful in protecting mice infected with erosols of influenz (A2/ Tiwn/64) nd prinfluenz type 1 (Sendi) viruses. Administrtion of poly I:C s n erosol offers n dvntge, prticulrly in therpy, by eliminting the risk of pulmonry dissemintion of virl infections due to intrnslly instilled fluids. Tretment of mice with erosols of poly I:C reduced the infection rte with influenz virus but did not inhibit virus multipliction in the lungs of most of those nimls where infection becme estblished. Sendi virus infection rtes were undiminished in mice treted with poly I:C, but lung-virus titers were significntly suppressed s compred with those of untreted nimls. The mximum poly I:C doses (40,g) dministered by erosol produced no evidence of toxicity in the mice. The discovery of interferon induction by double-strnded complexes of polyinosinic nd polycytidylic cids (poly I :C; reference 2) hs led to severl reports demonstrting the effectiveness of this inducer in protecting lbortory hosts from virl infections (2, 7-10, D. A. Hill nd S. Bron, Bcteriol. Proc., p. 149, 1969). Although poly I:C hs been dministered to nimls by intrnsl, intrperitonel, nd intrvenous inocultions or by oculr instilltion, there hve been no reports of erosol dministrtion. Intrnsl dministrtion of poly I:C prior to infection protects mice ginst intrnsl chllenge with influenz virus. However, tretment fter infection hs not been effective (unpublished observtions). This my be due to the known dissemintion nd enhncement of influenz virus infection by fluids dministered intrnslly fter infection (13). Since erosol dministrtion my not disseminte virus, this method my ultimtely be useful in the therpy of influenz infections. A study ws therefore mde of the efficcy of irborne dministrtion of poly I:C during myxovirus infection of mice s guide to its possible usefulness in therpeutic studies. The following communiction describes the effectiveness of poly I:C dministered in erosol 1 Present ddress: 75 Edwrdel Rod, Needhm, Mss Present ddress: nd Avenue, N.E., Bellevue, Wsh form to mice in protecting ginst infections with influenz (A2/Tiwn/64) nd prinfluenz type 1 (Sendi) viruses. Continution of poly I:C tretment up to the third dy postinfection did not pper to enhnce the virl infection nor overcome the protective effect of previous tretment. MATERIALS AND METHODS Poly I:C. The synthetic double-strnded poly I:C used in these studies ws prepred by mixing equimolr concentrtions of poly I nd poly C (P. L. Biochemicls, Inc.). The single-strnded homopolymers hd moleculr weight of pproximtely 105 dltons. The finl concentrtion of poly I:C ws 1.0 mg/ml in physiologicl sline. Mice. All mice used in these experiments were mles of the Swiss-Webster strin produced t the Fort Detrick Animl Frm. The mice were in the 16- to 20-g rnge t the beginning of ech experiment. Viruses. The influenz virus used in these experiments ws the A2/Tiwn/64 strin. The virus ws propgted in 10-dy-old embryonted hens' eggs tht were inoculted by the choriollntoic route nd incubted for 48 hr t 35 C before the llntoic fluid ws hrvested. The working seeds contined medin egg infectious doses (E1D50) per milliliter. The Sendi strin of prinfluenz virus type 1 ws obtined from the Reserch Reference Regents Brnch of the Ntionl Institutes of Helth. For these experiments, virus ws produced in embryonted eggs (third pssge) s described bove for influenz, 323

2 324 GERONE ET AL. INFEC. IMMUN. except tht the infected eggs were incubted for 72 hr insted of 48 hr. The Sendi virus pool contined 109" E1D50 per ml. Virus ssy. Both the Sendi nd influenz viruses were ssyed by injecting 0.1 ml of virus dilution into 10-dy-old embryonted eggs by the choriollntoic route. After 72 hr of incubtion, the llntoic fluids were hrvested nd individully tested for hemgglutinin by using chicken erythrocytes s n indictor of infection. The EID50's were clculted by the Krber method (6). Exposure of mice to virl erosols. Mice were exposed to virl erosols generted with Collison tomizer in modified Henderson pprtus (5). Sendi virus ws diluted 10-3 in beef hert infusion broth (BHIB) nd the influenz virus ws tomized either s undiluted llntoic fluid or s 10-1 dilution in BHIB. The mice were exposed to the dynmic erosols for 3-min period. An ll-glss impinger contining 10 ml of BHIB ws used to smple the virus erosols for 1 min t the midpoint of the niml exposure. The virus concentrtion in the impinger fluid ws used to determine the erosol concentrtion. The inhled dose of the mice ws clculted by multiplying the erosol concentrtion per unit volume by the length of the exposure period nd by the brething rte (per minute) of the mice (4). Exposure of mice to erosols of poly I:C. Mice were treted with sttic erosols of poly I:C in 90- liter rotting drum (3). Ten to 20 mice were plced in ech of four rectngulr wire mesh bskets suspended in the interior of the erosol drum. The totl time exposure to erosol for ech tretment ws 1 hr. At the beginning of the exposure period nd t 15-min intervls therefter 4 ml of poly I:C (1 mg/ml, totl of 16 ml) ws erosolized with University of Chicgo Toxicologicl Lbortory glss tomizer (11). Using sodium fluorescein in the poly I:C solution s trcer for erosol recovery dt, it ws determined tht the mximum inhled dose per mouse per tretment did not exceed 8,ug of poly I:C. Serology. Infection of mice with either virus ws determined by the ppernce of hemgglutintinginhibiting (HI) ntibodies in plsm 3 to 4 weeks fter virus inocultion. The plsm ws collected from retro-orbitl blood vessels in heprinized micropipettes. The HI tests were performed by the microtiter technique (12) using 2 to 4 units of ntigen. A proportion of mice were bled before ech experiment nd were found to be invribly negtive for HI ntibody. RESULTS Susceptibility of poly I: C-treted mice to influenz virus. In two experiments, mice were treted with erosols of poly I:C 24 hr before exposure to erosols of A2/Tiwn/64 virus. The doses of virus used in these experiments infected 73% of the control, untreted mice (Tble 1). The single tretment with poly I:C reduced the overll infection rte to 32%. The geometric men titer of HI ntibody in plsm ws found to be somewht lower in the treted nimls in one experiment (PIC-1), but the reverse ws found in the second experiment (PIC-3). The protective effect of poly I:C could not be mesured in terms of reduced mortlity becuse it ws imprcticl to chieve high enough doses of virus by erosol dministrtion. In subsequent experiment mice were given three tretments with poly I:C. The first ws given 3 hr before dministrtion of virus, nd the second nd third tretments were given 24 nd 48 hr fter the virus. Ten mice from the control nd 10 mice from the treted groups tht received the lrger virus dose were scrificed t 72 hr for ssy of virus in lungs. The remining mice were held for 4 weeks nd then tested for the presence of HI ntibody in plsm. The infection rtes (Tble 2) of the mice were similr to those of the previous experiments (Tble 1). The geometric men titers of the treted nimls were only slightly lower thn those of the controls. The virus titers of the lungs (Tble 3) re similr in the control nd treted groups except for three mice in the treted group tht showed little or no virus (mice numbers 3, 5, nd 9). The men log virus titer of the remining mice in the treted group ws 4.10 EID50 compred with 4.35 EID50 for the controls. The men log virus titer of ll the treted mice ws 3.09 EID50. Effect of poly I:C tretment on Sendi virus infections of mice. Two groups of mice (A nd B) were treted with n erosol of poly I:C on 2 consecutive dys. After the second tretment, these mice nd two untreted control groups TABLE 1. Susceptibility of mice treted erosols of poly I: C to chllenge with irborne influenz virus with %Q Controlsb Poly I: C treted' Expt no. Is : 4)... cou 4.,-c _J GMT" i GMT 0 0 PIC / / PIC / / PIC / / PIC-3 7 9/ / Inhled dose, medin egg infectious dose. b Totl infected/totl no. of mice = 43/59 = 73%. c Exposure to poly 1:C preceded virus inocultion by 24 hr. Totl infected/totl no. of mice = 18/57 = 32%. d Bsed on seroconversion by hemgglutintinginhibiting (HI) test. e Geometric (log2) men HI titer of positive plsms.

3 VOL. 3, 1971 TREATMENT WITH AEROSOLS OF POLY I:C 325 TABLE 2. Effect of multiple tretments of mice with poly I: C on susceptibility to influenz virus Expt no. 3 It Controlsb Poly I: C tretedc th(d GMTe Z GMT (U0 PIC / / PIC / / Inhled dose, medin egg infectious dose (EID5o). b Totl infected/totl no. of mice = 24/30 = 80%/O. c Tretments with poly l:c were given t -3, 24, nd 48 hr reltive to virus inocultion. Totl infected/totl no. of mice = 15/30 = 50%6. d Infected bsed on seroconversion by hemgglutinting-inhibiting (HI) test. e Geometric (log2) men HI titer of positive plsms. TABLE 3. Effect of multiple tretmenits of mice with poly I: C ont inifluenz virus titers in lungs Mouse no S Men logc Log EIDso/0.1 ml of 10% lung' Controls Poly I:C tretedb < < or < Virus titers of lungs 72 hr fter inocultion. EID5o, medin egg infectious dose. b Tretments with poly L:C were given t -3, 24, nd 48 hr reltive to virus inocultion. c F-vlue is 3.65 for 1,18 degrees of freedom, suggesting tht the mens re significntly different t the 5% level. (C nd D) were exposed to Sendi virus erosols (77 to 194 EID50). Groups A nd B received dditionl tretments with poly I:C on dys 1, 2, nd 3 fter virus inocultion. Five mice were scrificed from ech of the groups on dy 1 nd gin on dy 3. Lungs were removed for virus ssy. The remining mice from ech group were bled t 14 dys nd gin t 23 dys for HI determintions on the plsms. At 24 hr none of the lungs from the groups treted with poly I:C hd virus (Tble 4). The men log titers of the untreted control groups were 2.64 nd 1.90 EID50. At 72 hr most of the lungs in the treted groups hd virus, but the men log titers were t lest 1,000-fold lower thn those of the untreted controls. The HI response of treted mice ws lower thn tht of controls, s seen by the geometric men titers of plsms collected t 14 dys (Tble 5). By the 23rd dy, however, there ppered to be no differences between the control nd the treted groups. Effect of vrious poly I: C tretment regimens on Sendi infections in mice. Six groups of ten mice ech were treted with poly I: C on vrious schedules. Group A ws treted on dys -1, 0, nd 1. Group B ws treted on dys -1 nd 0. Group C ws treted on dys 0 nd 1. Groups D, E, nd F were given single tretments on dys -1, 0, nd 1, respectively. Group G ws not treted. On dy 0, ll mice were exposed to n erosol dose of 95 EID50 of Sendi virus. All mice were scrificed 72 hr fter virus inocultion, nd lungs were exmined for virus content. The results in Tble 6 showed tht ll tretment groups except group F differed significntly from the control. Tretment groups A, B, nd C were significntly different from D, E, nd F. The TABLE 4. Effect of multiple tretments of mice with poly I: C on Sendi virus titers in lungs Logio EIDso/0.1 ml of 10% lung Time5 of lung MOuse Poly I: C tre~ted Control groups hrvest (hr) no. groupsc A B C D 24 1 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Men log > Neg Men log > Mice in groups A nd C received n inhled dose of 77 medin egg infectious doses (EID5o); mice in groups B nd D received 194 EID5. b Reltive to virus inocultion. c Mice treted on dys -1, 0, 1, 2, nd 3 reltive to virus inocultion.

4 326 GERONE ET AL. INFEC. IMMUN. sttisticl nlysis ws done by the Duncn multiple F test method (1). DISCUSSION The present study ws undertken to determine whether erosols of poly I:C could be used to protect mice infected with respirtory viruses. Although instilltion of poly I:C hs been reported to protect mice chllenged with influenz viruses (7), dministrtion of fluids could tend to disseminte nd enhnce pulmonry infections (13). The finding tht irborne dministrtion of n interferon inducer could be protective ginst influenz nd Sendi virus infections of mice, even though smll doses of poly I:C were given, TABLE 5. Effect of poiy I: C tretmiienit onz henmgglutinting-inl/ibitinig (HI) responise of mice exposed to Sendi virius Dys fter oli:c No. virus Group tretmenth infected per GMT inocultion temnt totlc 14 A Yes 9/ B Yes 9/ C No 11/ D No 12/ A Yes 9/ B Yes 10/ C No 10/ D No 11/12 5 Mice in groups A nd C received n inhled dose of 77 medin egg infectious doses (ElDo); mice in groups B nd D received 194 EID50 I Mice were treted on dys - 1, 0, 1, 2, nd 3 reltive to virus inocultion. c Bsed on seroconversion by HI test. d Geometric (log,) men HI titer. indictes tht this method of dministrtion holds promise. It might be predicted tht lrger doses of poly I: C given by the irborne route would increse the protective effect. Doses of poly I :C tht provide stronger interferon response would be required to chieve tretment of n lredy estblished infection (M. Worthington nd S. Bron, personl communiction). The dministrtion of poly I:C to mice by the erosol route effectively ltered their responses to influenz nd Sendi virus infections. The dt suggest tht the type of protection elicited ginst influenz virus ws somewht different from the protection observed when the nimls were chllenged with Sendi virus. In the cse of influenz the poly I:C tretments ppered to prevent infection in some of the inoculted nimls. Among treted mice tht becme infected, the virus titers of the lungs were no lower thn those in the untreted groups. On the other hnd, the poly I:C tretment did not lter the infection rtes of mice exposed to Sendi virus but did mrkedly reduce virus titers in their lungs t 24 nd 72 hr postinfection. Additionl experimenttion will be necessry to determine whether or not these differences re relted to the biologicl chrcteristics of the viruses or to the reltive doses employed in the virl chllenges. The best protection ginst Sendi virus infection ws observed when the poly I:C tretments were given before or on the dy of virus inocultion. Mice treted with poly I:C 24 hr fter exposure to the virus showed no significnt decreses in virus titers of the lungs compred to controls. The doses of poly I:C dministered in these experiments were only frction of those em- TABLE 6. Effect of vrt)iouls poly I: C tretmnent reginmenis oni linsg virlis titers of mice inifected wvith Senidci Mouse no. A B n(1,o)b (-1, c D E F 0) G (0,1) (-1) (0) (1) (none) 1 Neg 1.9c Neg Neg Neg Men logd Averge inhled dose ws 95 medin egg infectious doses (EID5e). bdys of poly I:C tretment reltive to virus inocultion. c Log,oEID50 per 0.1 ml of 10% lung suspension. d Mens A, B, nd C, C nd D, E nd F, D nd E, nd F nd G re not significntly different t the 5% level. Other combintions of mens re significntly different t the 1'- level.

5 VOL. 3, 1971 TREATMENT WITH AEROSOLS OF POLY I:C 327 ployed by other investigtors. A single erosol tretment consisted of mximum of 8 ug, nd the longest tretments were continued for 5 consecutive dys (40 jg, totl). Since these doses were clculted from the concentrtion of poly I:C in the erosols nd respirtory volumes of the mouse, the ctul dose retined by the mice my be considerbly less thn 8,ug. No evidence of toxicity ws seen in mice given 5-dy erosol tretment with poly I:C. Mice from the treted nd untreted groups were scrificed dily, nd lung tissues were prepred for histopthologicl exmintion. Histologic ltertions were not seen in poly I:C treted or untreted nimls. LITERATURE CITED 1. Duncn, D. B Multiple rnge nd multiple F tests. Biometrics 11: Field, A. K., A. A. Tytell, G. P. Lmpson, nd M. R. Hillemn Inducers of interferon nd host resistnce. H. Multistrnded synthetic polynucleotide complexes. Proc. Nt. Acd. Sci. U. S. A. 58: Goldberg, L. J., H. M. S. Wtkins, E. E. Boerke, nd M. A. Chtigny The use of rotting drum for the study of erosols over extended periods of time. Amer. J. Hyg. 65: Guyton, A. C Mesurement of the respirtory volume of lbortory nimls. Amer. J. Physiol. 150: Henderson, D. D An pprtus for the study of irborne infection. J. Hyg. 50: Lennette, E. H Generl principles underlying lbortory dignosis of virl nd rickettsil infections, p. 48. In E. H. Lennette nd J. J. Schmidt (ed.), Dignostic procedures for virl nd rickettsil diseses. Amer. Public Helth Ass., New York. 7. Lindh, H. F., H. L. Lindsy, B. R. Myberry, nd M. Forbes Polyinosinic-cytidylic cid complex (poly I:C) nd virl infections in mice. Proc. Soc. Exp. Biol. Med. 132: Nemes, M. M., A. A. Tytell, G. P. Lmpson, A. K. Field, nd M. R. Hillemn Inducers of interferon nd host resistnce. VI. Antivirl efficcy of poly I:C in niml models. Proc. Soc. Exp. Biol. Med. 132: Prk, J. H., nd S. Bron Herpetic kertoconjunctivitis: therpy with synthetic double-strnded RNA. Science 162: Richmond, J. Y., nd L. D. Hmilton Foot-nd-mouth disese virus inhibition induced in mice by synthetic doublestrnded RNA (polyriboinosinic nd polyribocytidylic cids). Proc. Nt. Acd. Sci. U. S. A. 64: Rosebury, T Experimentl ir-borne infection, p. 80. In Microbiologicl monogrphs. Willims nd Wilkins Co., Bltimore. 12. Sever, J. L Appliction of microtechnique to virl serologicl investigtions. J. Immunol. 88: Tylor, R. M Experimentl infection with influenz A virus in mice. J. Exp. Med. 73: Downloded from on Mrch 15, 2019 by guest